Methods and devices for preventing viral transmission

ABSTRACT

An apparatus includes multiple first reservoirs and multiple second reservoirs joined with a substrate. Selected ones of the multiple first reservoirs include a reducing agent, and first reservoir surfaces of selected ones of the multiple first reservoirs are proximate to a first substrate surface. Selected ones of the multiple second reservoirs include an oxidizing agent, and second reservoir surfaces of selected ones of the multiple second reservoirs are proximate to the first substrate surface.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application Ser. Nos. 62/987,786 filed Mar. 10, 2020;62/988,739 filed Mar. 12, 2020; and 63/012,370 filed Apr. 20, 2020. Theentire contents of each of these applications are incorporated byreference herein.

FIELD

Living organisms are affected by electrical stimulus. Accordingly,apparatus and techniques for applying electric stimulus to tissue havebeen developed to address a number of medical issues. The presentSpecification relates to methods and devices useful for preventing viraltransmission and infection.

BACKGROUND

An infectious organism can use a person's body to sustain itself,reproduce, and colonize. These infectious organisms are known aspathogens. Examples of pathogens include bacteria, viruses, fungi, andprions. Pathogens can multiply and adapt quickly. Some infections aremild and barely noticeable, but others are severe and life-threatening,and some are resistant to treatment. Infection can be transmitted in avariety of ways. These include skin contact, bodily fluids, contact withfeces, airborne particles, and touching an object that an infectedperson has also touched. How an infection spreads and its effect on thehuman body depend on the type of agent. The immune system is aneffective barrier against infectious agents, but colonies of pathogensmay grow too large for the immune system to fight. At this stage,infections can become harmful. Due to the severity of viral infections,preventing transmission of a virus can be a critical step in avoidinginfection. However, current filtration capabilities limit the antiviralprotection that masks can provide.

SUMMARY

Aspects disclosed herein include systems, devices, and methods forpreventing viral transmission or acquisition, for example usingbioelectric devices that comprise a multi-array matrix of biocompatiblemicrocells in the form of a gaiter, scarf, balaclava, shemagh, mask,such as a medical mask, a surgical mask, a respirator, or an insertshaped to fit within or without a gaiter, mask or respirator, forexample as a layer in a laminate construction, in a filter port, oraffixed to the mask, surgical hood or respirator. Embodiments comprisesubstrates comprising a multi-array matrix of biocompatible microcellsthat can form a mask or gaiter, or be inserted into a port or slot, oraffixed to a surgical mask or gaiter, for example the interior (facingthe user) side of a surgical mask or gaiter, the exterior side, or both,thereby providing antiviral properties to a mask or gaiter. Embodimentscomprise a system comprising a reusable surgical gaiter or mask andgaiter or mask inserts comprising substrates comprising a multi-arraymatrix of biocompatible microcells. Disclosed systems can comprise aninsert comprising a hydration layer comprising a conductive solution toestablish and maintain microcurrents. Disclosed inserts can bereversibly attached to a reusable gaiter or mask. In this manner, agaiter or mask can be “recharged” with antiviral properties.

Embodiments comprise a system comprising inserts comprising substratescomprising a multi-array matrix of biocompatible microcells. Disclosedsystems can comprise an insert comprising a hydration layer comprising aconductive solution to establish and maintain microcurrents.

Disclosed embodiments comprise washable gaiters, gaiter inserts, masksand mask inserts that can be re-used.

Disclosed embodiments combine filtration with further antiviralproperties.

Aspects disclosed herein comprise bioelectric devices comprising amulti-array matrix of biocompatible microcells. Such matrices caninclude a first array comprising a pattern of microcells, for exampleformed from a first conductive solution, the solution including a metalspecies; and a second array comprising a pattern of microcells, forexample formed from a second conductive solution, the solution includinga metal species capable of defining at least one voltaic cell forspontaneously generating at least one electrical current with the metalspecies of the first array when said first and second arrays areintroduced to an electrolytic solution such as saline, and said firstand second arrays are not in physical contact with each other. Certainaspects utilize an external power source such as AC or DC power orpulsed RF or pulsed current, such as high voltage pulsed current. In oneembodiment, the electrical energy is derived from the dissimilar metalscreating a battery at each cell/cell interface, whereas thoseembodiments with an external power source may employ conductiveelectrodes in a spaced apart configuration to predetermine the electricfield shape and strength. The external source can provide energy for alonger period than the batteries on the surface.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a detailed plan view of an embodiment disclosed herein.

FIG. 2 is a detailed plan view of a pattern of applied electricalconductors in accordance with an embodiment disclosed herein.

FIG. 3 is an embodiment using the applied pattern of FIG. 2.

FIG. 4 is a cross-section of FIG. 3 through line 3-3.

FIG. 5 is a detailed plan view of an embodiment disclosed herein whichincludes fine lines of conductive metal solution connecting electrodes.

FIG. 6 is a detailed plan view of an embodiment having a line patternand dot pattern.

FIG. 7 is a detailed plan view of an embodiment having two linepatterns.

FIG. 8A depicts an embodiment showing the location of discontinuousregions as well as anchor regions of the system.

FIG. 8B depicts an embodiment showing the location of discontinuousregions as well as anchor regions of the system.

FIG. 8C depicts an embodiment showing the location of discontinuousregions as well as anchor regions of the system.

FIG. 8D depicts an embodiment showing the location of discontinuousregions as well as anchor regions of the system.

FIG. 8E depicts an embodiment showing the location of discontinuousregions as well as anchor regions of the system.

FIG. 9 depicts an embodiment showing a mask comprising a multi-arraymatrix of biocompatible microcells and means for securing the mask.

FIG. 10 depicts an embodiment showing a mask.

FIG. 11 depicts a substrate described herein.

FIG. 12 depicts PROCELLERA® (an embodiment disclosed herein) output overtime.

FIG. 13 depicts an embodiment showing a mask.

FIG. 14(A) is an Energy Dispersive X-ray Spectroscopy (EDS) analysis ofAg/Zn BED (“bioelectric device” refers to an embodiment as disclosedherein).

-   -   a. Scanning Electron Microscope (SEM) image;    -   b. Light Microscope Image;    -   c. Closer view of a golden dot and a grey dot in B respectively.    -   d. Closer view of a golden dot and a grey dot in B respectively.    -   e. EDS element map of zinc;    -   f. EDS element map of silver;    -   g. EDS element map of oxygen;    -   h. EDS element map of carbon. Scale bar A-B, E-H: 1 mm; C-D: 250        μm    -   14(B, C) Absorbance measurement on treating planktonic PAO1        culture with placebo, Ag/Zn BED and placebo+Ag dressing; and CFU        measurement.

14(D) Zone of inhibition with placebo, Ag/Zn BED and placebo+Agdressing.

FIG. 15 depicts Scanning Electron Microscope (SEM) images of in-vitroPseudomonas aeruginosa PAO1 biofilm treated with placebo, an embodimentdisclosed herein (“BED”), and placebo+Ag dressing. The BED treatedbiofilm shows a dramatic decrease in bacteria number.

FIG. 16 shows extracellular polysaccharide staining (EPS).

FIG. 17 shows live/dead staining. The green fluorescence indicates livePAO1 bacteria while the red fluorescence indicates dead bacteria.

FIG. 18A shows PAO1 staining of the biofilm demonstrating the lack ofelevated mushroom like structures in the Ag/Zn BED treated sample. FIG.18B shows PAO1 staining of the biofilm demonstrating the lack ofelevated mushroom like structures in the Ag/Zn BED treated sample. FIG.18C shows PAO1 staining of the biofilm demonstrating the lack ofelevated mushroom like structures in the Ag/Zn BED treated sample.

FIG. 19 depicts real-time PCR to assess quorum sensing gene expression.

FIG. 20A shows electron paramagnetic (EPR) spectra using DEPMPO (aphosphorylated derivative of the widely used DMPO spin trap) for aplacebo. Spin adduct generation upon exposure to disclosed embodimentsfor 40 minutes in PBS. FIG. 20B shows electron paramagnetic (EPR)spectra using DEPMPO (a phosphorylated derivative of the widely usedDMPO spin trap) for a disclosed embodiment.

FIG. 21 depicts real-time PCR performed to assess mex gene expressionupon treatment with Ag/Zn BED and 10 mM DTT.

FIG. 22A shows Glycerol-3-Phosphate Dehydrogenase (GPDH) enzymeactivity.

-   -   a. In FIG. 22A, OD was measured in the kinetic mode.    -   b. In FIG. 22B, GPDH activity was calculated using the formula,        Glycerol-3-Phosphate dehydrogenase activity=B/(ΔT×V)×Dilution        Factor=nmol/min/ml, where: B=NADH amount from Standard Curve        (nmol). ΔT=reaction time (min). V=sample volume added into the        reaction well (ml).

FIG. 23 depicts a mask embodiment comprising pleats and a bendableportion atop the mask to allow the user to better conform the mask toher face.

FIG. 24 depicts a mask embodiment comprising pleats and a bendableportion atop the mask to allow the user to better conform the mask toher face.

FIG. 25 depicts a mask embodiment comprising a “snap” closure.

FIG. 26 depicts a mask embodiment.

FIG. 27A shows a negative control plate with no antiviral treatment.FIG. 27B shows the effect of a zinc treatment alone. FIG. 27C shows theeffect of a silver treatment alone. FIG. 27D shows the antiviral effectof a disclosed embodiment as described in Example 16.

FIG. 28A shows a negative control plate with no antiviral treatment.FIG. 28B shows the effect of a zinc treatment alone. FIG. 28C shows theeffect of a silver treatment alone. FIG. 28D shows the antiviral effectof a disclosed embodiment as described in Example 16.

FIG. 29A depicts quantification of the purified viral particles afterspotting on fe (an embodiment disclosed herein) yielded 44.29% and23.73% recovery from the fabric when exposed for 1 min or 5 min,respectively. Nanoparticle tracking analysis demonstrated that unlikethe purified CoV that showed a single peak around 75 nm, the recoveredCoV showed additional peaks suggesting aggregation of the viralparticles upon contact with the fabric (FIG. 29B). Analysis of zetapotential showed significant graded attenuation of this electrokineticproperty upon contact with the fe (FIG. 29C). Such lowering of averagezeta potential of CoV, applied and recovered from fe, has been plottedgraphically (FIG. 29D). FIG. 29E shows the potential distribution.Unlike 1 min exposure to the fe, 5 min exposure showed an appreciabledifference in the phase plot of the viral particles (FIG. 29F).

FIG. 30A shows cells treated with fe-recovered CoV particles appeared ashealthy as the uninfected cells; To assess changes in the infectivity ofCoV following contact with the electroceutical fabric, a cytopathicassay was employed. Infected cells were monitored for appearance ofcytopathic effects (CPE; cell rounding and sloughing) untilpost-infection day 7. Overt CPE was observed on day 7 in response to CoVinfection (FIG. 30B). Comparable CPE was noted in response to treatmentof cells with CoV recovered from sham control fabric fs (FIG. 30C). Incontrast, CoV recovered from fe did not cause any CPE indicating loss ofits infectivity (FIG. 30D). Objective assessment of cell viability wasperformed using a calcein/PI fluorescence assay. Only live cells withintracellular esterase activity hydrolyze the acetoxymethyl ester innon-fluorescent Calcein AM converting it into green fluorescent Calcein.Dead cells or cells with damaged or compromised cell membranes includePI stain, which is otherwise impermeant to live cells. Fold-changeincrease in PI/Calcein signal as shown indicates loss of cell viabilityin response to infection. Infection of cells with CoV caused marked lossof cell viability (FIG. 30B). Such cytopathic effect of CoV wascompletely absent once the virus was exposed to fe (FIGS. 30 D-E). Thesham fabric did not afford such protection (FIGS. 30C,E). The cytopathiceffects of CoV and the protective effects of fe (versus fs) wascorroborated by the standard MTT assay commonly used for testing cellviability (FIG. 30F).

FIG. 31A; The Lentiviral pseudotype system is a standard laboratory toolto study the infectivity of viruses under conventional biosafetyconditions. Lentivirus CSCGW mut6, upon successful transduction inHEK293 cells, results in GFP-expressing host cells. This expression is adirect measure of lentiviral replication competency and ability of thevirus to integrate in the host genome. The ability of theelectroceutical fabric to influence the infectivity of a virus, otherthan CoV, was tested to appreciate its broader significance of scope.Mammalian cells were treated with purified lentivirus or the same virussubjected to contact with fe or fs for 1 or 5 mins as indicated in thefigure legend (FIG. 31A). Transduced cells were monitoredmicroscopically to check the presence of GFP+ cells, a marker ofsuccessful infection. Lentiviral exposure caused widespread infection ofcells. Treatment of cells with virus recovered from sham fabric fscaused comparable infection (FIG. 31B). However, contact of virus withthe electroceutical fabric fe, even for one minute, eliminatedlentiviral infectivity (FIG. 31B).

FIG. 32A shows SEM of the fabric used for a disclosed mask showed adifferent weaving pattern aimed at higher stretch property. FIG. 32Bshows SEM of the fabric used for a disclosed mask showed a differentweaving pattern aimed at higher stretch property. FIG. 32C shows SEM ofthe fabric used for a disclosed mask showed a different weaving patternaimed at higher stretch property. Deposition of Ag and Zn on the fabricfor face-mask was tested by EDX spectrum analysis (FIG. 32B).

FIG. 33; To assess changes in the infectivity of CoV following contactwith the electroceutical fabric, a cytopathic assay was employed.Infected cells were monitored for appearance of cytopathic effects (CPE;cell rounding and sloughing) until post-infection day 7. Overt CPE wasobserved on day 7 in response to CoV infection. Comparable CPE was notedin response to treatment of cells with CoV recovered from sham controlfabric fs. In contrast, CoV recovered from fe did not cause any CPEindicating loss of its infectivity. Cells treated with fe-recovered CoVparticles appeared as healthy as the uninfected cells. Objectiveassessment of cell viability was performed using a calcein/PIfluorescence assay. Only live cells with intracellular esterase activityhydrolyze the acetoxymethyl ester in non-fluorescent Calcein AMconverting it into green fluorescent Calcein. Dead cells or cells withdamaged or compromised cell membranes include PI stain, which isotherwise impermeant to live cells. Fold-change increase in PI/Calceinsignal as shown indicates loss of cell viability in response toinfection. Infection of cells with CoV caused marked loss of cellviability. Such cytopathic effect of CoV was completely absent once thevirus was exposed to fe. The sham fabric did not afford such protection.The cytopathic effects of CoV and the protective effects of fe (versusfs) was corroborated by the standard MTT assay commonly used for testingcell viability.

FIG. 34 shows the front of a disclosed gaiter embodiment, withelectrodes visible on the outside of the device and ear loops.

FIG. 35 shows the back of a disclosed gaiter embodiment, with electrodesvisible on the outside of the device and ear loops.

FIG. 36 shows the back of a disclosed gaiter embodiment, with electrodesvisible on the outside of the device, Velcro to secure the device, andear loops.

FIG. 37 shows the side of a disclosed gaiter embodiment, with electrodesvisible on the outside of the device and ear loops.

FIG. 38 shows the front of a disclosed gaiter embodiment, withelectrodes visible on the outside of the device and ear loops.

FIG. 39 shows the inside front of a disclosed gaiter embodiment, withelectrodes visible on the outside of the device and ear loops.

FIG. 40 shows the device folding into an inner pouch forstorage/transport.

FIG. 41 shows the device folded into an inner pouch forstorage/transport.

DETAILED DESCRIPTION

Embodiments disclosed herein include systems that can provide a lowlevel electric field (LLEF) to a location, tissue or organism (an “LLEFsystem”) or, when brought into contact with an electrically conductingmaterial, can provide a low level electric current (LLEC) to a location,environment, tissue or organism (an “LLEC system”), thereby limiting orpreventing viral transmission. Thus, in embodiments an LLEC system is anLLEF system that is in contact with an electrically conducting material,for example saline, for example saline applied by a user prior to use ofthe system. In certain embodiments, the electric current or electricfield can be modulated, for example, to alter the duration, size, shape,field depth, duration, current, polarity, or voltage of the system. Inembodiments the watt-density of the system can be modulated. Poweredsystems are also disclosed herein.

“Activation gel” or “activation liquid” as used herein means acomposition useful for maintaining a moist environment within and aboutthe device. Activation gels can be conductive. In embodiments, the watervapor in a user's breath can act as an activation gel or liquid. Salinecan act as an activation gel or liquid.

“Affixing” as used herein can mean contacting a device, patient ortissue with a device or system disclosed herein. In embodiments“affixing” can include the use of snaps, loops, clips, straps, elastic,etc., and combinations thereof to join (reversibly or non-) the gaiterto a treatment area, or to a “port” or receiving portion of the gaiter,or to connect the opposite sides of the long axis of the gaiter to oneanother.

“Applied” or “apply” as used herein refers to contacting a surface witha conductive material, for example printing, painting, or spraying aconductive ink on a surface. Alternatively, “applying” can meancontacting a patient or tissue or organism with a device or systemdisclosed herein.

“Conductive material” as used herein refers to an object or type ofmaterial which permits the flow of electric charges in one or moredirections. Conductive materials can include solids such as metals orcarbon, or liquids such as conductive metal solutions and conductivegels. Conductive materials can be applied to form at least one matrix.Conductive liquids can dry, cure, or harden after application to form asolid material.

“Discontinuous region” as used herein refers to a “void” in a materialsuch as a hole, slit, slot, or the like. The term can mean any void inthe material though typically the void is of a regular shape. A void inthe material can be entirely within the perimeter of a material or itcan extend to the perimeter of a material.

“Dots” as used herein refers to discrete deposits of dissimilarreservoirs that can function as at least one battery cell. The term canrefer to a deposit of any suitable size or shape, such as squares,circles, triangles, lines, etc. The term can be used synonymously withmicrocells, electrodes, etc.

“Electrode” refers to similar or dissimilar conductive materials. Inembodiments utilizing an external power source the electrodes cancomprise similar conductive materials. In embodiments that do not use anexternal power source, the electrodes can comprise dissimilar conductivematerials that can define an anode and a cathode.

“Expandable” as used herein refers to the ability to stretch whileretaining structural integrity and not tearing. The term can refer tosolid regions as well as discontinuous or void regions; solid regions aswell as void regions can stretch or expand.

“Insert” (used as a noun) or “gaiter insert” refers to a disclosedembodiment that is designed to form, fit inside, or fit around a neckgaiter. The insert can comprise adhesive, snaps, Velcro, clips, or othermeans for securing it to a neck gaiter. For example, adhesive “flaps”can be employed to affix the insert or cover to the gaiter. The insertcan be placed into a pouch or slot in the neck gaiter to secure it tothe neck gaiter, for example an “envelope-slot” in the neck gaiterInserts can be an aspect of a disclosed system comprising a reusableneck gaiter and replaceable neck gaiter inserts.

Inserts or covers can comprise a polyester substrate.

“Matrix” or “matrices” as used herein refer to a pattern or patterns,such as those formed by electrodes on a surface, such as a fabric or afiber, or the like. Matrices can be designed to vary the electric fieldor electric current or microcurrent generated. For example, the strengthand shape of the field or current or microcurrent can be altered, or thematrices can be designed to produce an electric field(s) or current ormicrocurrent of a desired strength or shape.

“Stretchable” as used herein refers to the ability of embodiments thatstretch without losing their structural integrity. That is, embodimentscan stretch to accommodate irregular skin surfaces or surfaces whereinone portion of the surface can move relative to another portion.Stretchable embodiments can comprise, for example, elastic or acompression fabric.

“Viral transmission” refers to the transfer of a virus from one organismto another, for example via propagation of the virus from an infectedorganism or via acquisition of the virus by a healthy organism, orcombinations thereof.

LLEC/LLEF Systems and Devices

In embodiments, systems and devices disclosed herein comprise patternedmicro-batteries that create a unique field between each dot pair. Inembodiments, the unique field is very short, i.e. in the range of thephysiologic electric fields. In embodiments, the direction of theelectric field produced by devices disclosed herein is similar tophysiological conditions.

Embodiments disclosed herein can comprise patterns of microcells orelectrodes or reservoirs. The patterns can be designed to produce anelectric field, an electric current, or both over and through tissuesuch as human skin. In embodiments the pattern can be designed toproduce a specific size, strength, density, shape, or duration ofelectric field or electric current. In embodiments, electrode,reservoir, or microcell size and separation can be altered.

In embodiments devices disclosed herein can apply an electric field, anelectric current, or both, wherein the field, current, or both can be ofvarying size, strength, density, shape, or duration in different areasof the embodiment. In embodiments, by micro-sizing the electrodes orreservoirs, the shapes of the electric field, electric current, or bothcan be customized, increasing or decreasing very localized wattdensities and allowing for the design of “smart patterned electrodes”where the amount of electric field over a tissue can be designed orproduced or adjusted based on feedback from the tissue or on analgorithm within sensors operably connected to the embodiment andfed-back to a control module. The electric field, electric current, orboth can be strong in one zone and weaker in another. The electricfield, electric current, or both can change with time and be modulatedbased on treatment goals or feedback from the tissue or patient. Thecontrol module can monitor and adjust the size, strength, density,shape, or duration of electric field or electric current based on tissueparameters. For example, embodiments disclosed herein can produce andmaintain very localized electrical events. For example, embodimentsdisclosed herein can produce specific values for the electric fieldduration, electric field size, electric field shape, field depth,current, polarity, and/or voltage of the device or system.

Devices disclosed herein can generate a localized electric field in apattern determined by the size, distance between, and physicalorientation of the cells or electrodes. Effective depth of the electricfield can be predetermined by the orientation and distance between thecells or electrodes.

Embodiments of LLEC or LLEF systems disclosed herein can compriseelectrodes or microcells. Each electrode or microcell can be or includea conductive metal. In embodiments, the electrodes or microcells cancomprise any electrically-conductive material, for example, anelectrically conductive hydrogel, metals, electrolytes, superconductors,semiconductors, plasmas, and nonmetallic conductors such as graphite andconductive polymers. Electrically conductive metals can include silver,copper, gold, aluminum, molybdenum, zinc, lithium, tungsten, brass,carbon, nickel, iron, palladium, platinum, tin, bronze, carbon steel,lead, titanium, stainless steel, mercury, Fe/Cr alloys, and the like.The electrode can be coated or plated with a different metal such asaluminum, gold, platinum or silver.

In certain embodiments, dot, reservoir, or electrode geometry cancomprise circles, polygons, lines, zigzags, ovals, stars, or anysuitable variety of shapes. This provides the ability todesign/customize surface electric field shapes as well as depth ofpenetration. For example. In embodiments it can be desirable to employan electric field of greater strength or depth in an area where skin isthicker.

Reservoir or dot sizes and concentrations can be of various sizes, asthese variations can allow for changes in the properties of the electricfield created by embodiments of the invention. Certain embodimentsprovide an electric field at about 1 Volt and then, under normal tissueloads with resistance of 100k to 300K ohms, produce a current in therange of 1 to 10 microamperes.

A system disclosed herein and placed over tissue such as skin can moverelative to the tissue. Reducing the amount of motion between tissue anddevice can be advantageous to skin treatment. Slotting or placing cutsinto the device can result in less friction or tension on the skin. Inembodiments, use of an elastic dressing similar to the elasticity of theskin is disclosed. In embodiments, disclosed devices comprise a slotfrom around the center of the device that runs to an outside edge.

In embodiments the system comprises a component such as an adhesive,snaps, clips, or straps to maintain or help maintain its position. Forexample, an insert or cover can comprise a positioning component to“fix” its position relative to a neck gaiter, or a neck gaiter cancomprise a positioning component to fix the position of the insert orcover. The adhesive component can be covered with a protective layerthat is removed to expose the adhesive at the time of use. Inembodiments the adhesive can comprise, for example, sealants, such ashypoallergenic sealants, gecko sealants, mussel sealants, waterproofsealants such as epoxies, and the like. Straps can include Velcro orsimilar materials to aid in maintaining the position of the device.Straps can tie to other straps, for example encircling the head of auser. In embodiments, snaps can be used to secure the device, or tosecure an overlapping portion of the device to maintain a desired shape,for example the shape of a neck gaiter as seen in FIG. 34. Disclosedneck gaiters can comprise loops, for example elastic loops, to securethe device against the user's face (FIG. 34) and/or compression fabricsto secure the device in a desired position. In general, the compressionfabrics described herein may be configured to exert a suitable pressureof between about 2 mm Hg and about 10 mmHg to provide an elastic effect.In embodiments the pressure applied by the garment can be, for example,between 0 and 5 mmHg, or the like

Disclosed neck gaiters can comprise planar shapes wherein one end of theneck gaiter can be reversibly attached to an other end (for example atthe opposite ends of the long axis) to secure the device in a desiredposition. Disclosed reversible attachments can comprise hooks, snaps,magnets, Velcro, and the like.

In embodiments, the LLEC or LLEF system can comprise a laminate wherelayers of the laminate can be of varying elasticities. For example, anouter layer may be highly elastic and an inner layer in-elastic or lesselastic. The in-elastic layer can be made to stretch by placing stressrelieving discontinuous regions or slits through the thickness of thematerial so there is a mechanical displacement rather than stress thatwould break the fabric weave before stretching would occur. Inembodiments the slits can extend completely through a layer or thesystem or can be placed where expansion is required. In embodiments ofthe system the slits do not extend all the way through the system or aportion of the system such as the substrate.

In embodiments, disclosed devices can be shaped to fit an area ofdesired use, for example the human face, or any area where preventingviral transmission is desired. For example, disclosed embodimentscomprise neck gaiters, such as reusable neck gaiters, or neck gaiterinserts, comprising patterned micro-batteries that create a unique fieldbetween each dot pair.

Further embodiments comprise substrates shaped to fit inside or outsidea neck gaiters wherein the substrate, for example a planar or non-planarsubstrate, or a planar substrate that can fold into a non-planar form,comprises patterns of microcells. The substrate can be reversiblyattached to the mask. The substrate can comprise a hydration element,for example a hydration layer, or hydration reservoir. In embodimentscomprising reversibly attached inserts, the insert can be replaced aftera period of time. For example, the neck gaiter insert can be disengagedfrom the neck gaiter and replaced with another insert after 12 hours, 11hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3hours, 2 hours, 1 hour, or the like. In embodiments, the mask insert isreplaced after no more than 12 hours, no more than 11 hours, no morethan 10 hours, no more than 9 hours, no more than 8 hours, no more than7 hours, no more than 6 hours, no more than 5 hours, no more than 4hours, no more than 3 hours, no more than 2 hours, no more than 1 hour,or the like.

In embodiments, disclosed systems and elements thereof are washable. Forexample, in embodiments, a neck gaiter insert is removed after a periodof time, washed, and then reused.

Disclosed microcell patterns can be designed to produce an electricfield, an electric current, or both over and through tissue such ashuman skin. In embodiments the pattern can be designed to produce aspecific size, strength, density, shape, or duration of electric fieldor electric current. In embodiments reservoir or dot size and separationcan be altered. The planar substrate can further comprise adhesive. Forexample at the perimeter (or portion thereof) of the substrate, adhesivecan provide the user the ability to securely fasten the substrate insidea neck gaiter, outside a neck gaiter, or within a pouch or port in theneck gaiter.

In certain embodiments, for example methods of use, it can be preferableto utilize AC or DC current. For example, embodiments disclosed hereincan employ phased array, pulsed, square wave, sinusoidal, or other waveforms, or the like. Certain embodiments utilize a controller to produceand control power production and/or distribution to the device.

Embodiments disclosed herein comprise biocompatible electrodes orreservoirs or dots on a surface or substrate, for example a fabric, afiber, or the like. In embodiments the surface or substrate can bepliable, for example to better follow the contours of an area to betreated, such as the face. In embodiments the surface can comprise agauze or mesh or plastic. Suitable types of pliable surfaces for use inembodiments disclosed herein can be absorbent or non-absorbent textiles,low-adhesives, vapor permeable films, hydrocolloids, hydrogels,alginates, foams, foam-based materials, cellulose-based materialsincluding Kettenbach fibers, hollow tubes, fibrous materials, such asthose impregnated with anhydrous/hygroscopic materials, beads and thelike, or any suitable material as known in the art. In embodiments thepliable material can form, for example, a neck gaiter, such as that wornon the face, or the like, or an insert shaped to fit a neck gaiter.Embodiments can comprise multiple layers. Multi layer embodiments caninclude, for example, a skin-contacting layer, a layer comprisingmicrocells, and an outer layer.

Disclosed embodiments can comprise non-woven fabric, for examplepolypropylene. In embodiments, the polypropylene can have a density of,for example, 15, 20, 25, 30, 35, or more g/M². In further embodiments, amask can comprise, for example, polyester, cotton, microfiber, cupra,Tencel, bio-cellulose, charcoal, and the like.

Embodiments can include coatings on the surface, such as, for example,over or between the electrodes or cells. Such coatings can include, forexample, salts, antivirals, antibacterials, conductive fluid or gel, orthe like.

In embodiments the system comprises a component such as compressionfabric or elastic to maintain or help maintain its position. Inembodiments the system comprises components such as ear loops, snaps,Velcro, clips, or straps to maintain or help maintain its position. Incertain embodiments the system or device comprises a clip or strap, forexample on either end of the long axis, or a clip or strap linking onend of the long axis to the other. In embodiments that straps cancomprise Velcro or a similar fastening system. In embodiments the strapscan comprise elastic materials. In further embodiments the strap cancomprise a conductive material, for example a wire to electrically linkthe device with other components, such as monitoring equipment or apower source. In embodiments the device can be wirelessly linked tomonitoring or data collection equipment, for example linked viaBluetooth to a cell phone that collects data from the device. In certainembodiments the device can comprise data collection means, such astemperature, pressure, or conductivity data collection means. In certainembodiments the device can comprise data transmission means. Embodimentscan comprise a visible indicator to confirm that the insert or cover ishydrated, producing microcurrents, or both.

An LLEC or LLEF system disclosed herein can comprise straps to affix thesystem securely, for example around the head of a user. An LLEC or LLEFsystem disclosed herein can comprise adhesive to affix the systemsecurely, for example affix it inside a neck gaiter.

In embodiments, the LLEC or LLEF system can comprise instructions ordirections on how to place and use the system to maximize itsperformance. For example, disclosed kits can comprise instructionsregarding “wetting” the system with a conductive liquid prior to use.

Embodiments can comprise a kit comprising a device disclosed herein andan antiviral or antibacterial material. Disclosed kits con compriseinstructions for use.

Embodiments can comprise a system comprising a reusable mask and maskinserts that can be reversibly attached to the mask, for example to theinside of the mask, to the outside of the mask, within a slot or pouchof the mask, for example an envelope-type slot, or the like.

LLEC/LLEF Systems and Devices; Methods of Manufacture

In embodiments dissimilar metals can be used to create an electric fieldwith a desired voltage. In certain embodiments the pattern of reservoirscan control the watt density and shape of the electric field.

In embodiments printing devices can be used to produce LLEC or LLEFsystems disclosed herein. For example, inkjet or “3D” printers can beused to produce embodiments. In embodiments “ink” or “paint” cancomprise any conductive solution suitable for forming an electrode on asurface, such as a conductive metal solution. In embodiments “printing”or “painted” can comprise any method of applying a conductive materialsuch as a conductive liquid material to a material upon which a matrixis desired, such as a fabric.

In certain embodiments the binders or inks used to produce LLEC or LLEFsystems disclosed herein can include, for example, poly cellulose inks,poly acrylic inks, poly urethane inks, silicone inks, and the like. Inembodiments the type of ink used can determine the release rate ofelectrons from the reservoirs. In embodiments various materials can beadded to the ink or binder such as, for example, conductive or resistivematerials can be added to alter the shape or strength of the electricfield.

In embodiments, electroceutical fabric embodiments disclosed herein canbe woven or non-woven. For example, disclosed embodiments can be wovenof at least two types of fibers; fibers comprising sections treated orcoated with a substance capable of forming a positive electrode; andfibers comprising sections treated or coated with a substance capable offorming a negative electrode. The fabric can further comprise fibersthat do not form an electrode. Long lengths of fibers can be woventogether to form fabrics. For example, the fibers can be woven togetherto form a regular pattern of positive and negative electrodes.

Certain embodiments can utilize a power source to create the electriccurrent, such as a battery or a microbattery. The power source can beany energy source capable of generating a current in the system and caninclude, for example, AC power, DC power, radio frequencies (RF) such aspulsed RF, induction, ultrasound, and the like.

Dissimilar metals used to make an LLEC or LLEF system disclosed hereincan be silver and zinc, and the electrolytic solution can include sodiumchloride in water. In certain embodiments the electrodes are appliedonto a non-conductive surface to create a pattern, most preferably anarray or multi-array of voltaic cells that do not spontaneously reactuntil they contact an electrolytic solution. Sections of thisdescription use the terms “printing” with “ink,” but it is understoodthat the patterns may instead be “painted” with “paints.” The use of anysuitable means for applying a conductive material is contemplated. Inembodiments “ink” or “paint” can comprise any solution suitable forforming an electrode on a surface such as a conductive materialincluding a conductive metal solution. In embodiments “printing” or“painted” can comprise any method of applying a solution to a materialupon which a matrix is desired.

A preferred material to use in combination with silver to create thevoltaic cells or reservoirs of disclosed embodiments is zinc. Zinc hasbeen well-described for its uses in prevention of infection in suchtopical antibacterial agents as Bacitracin zinc, a zinc salt ofBacitracin. Zinc is a divalent cation with antibacterial properties ofits own.

Turning to the figures, in FIG. 1, the dissimilar first electrode 6 andsecond electrode 10 are applied onto a desired primary surface 2 of anarticle 4. In one embodiment a primary surface is a surface of an LLECor LLEF system that comes into direct contact with an area to be treatedsuch as a skin surface.

In various embodiments the difference of the standard potentials of theelectrodes or dots or reservoirs can be in a range from about 0.05 V toapproximately about 5.0 V. For example, the standard potential can beabout 0.05 V, about 0.06 V, about 0.07 V, about 0.08 V, about 0.09 V,about 0.1 V, about 0.2 V, about 0.3 V, about 0.4 V, about 0.5 V, about0.6 V, about 0.7 V, about 0.8 V, about 0.9 V, about 1.0 V, about 1.1 V,about 1.2 V, about 1.3 V, about 1.4 V, about 1.5 V, about 1.6 V, about1.7 V, about 1.8 V, about 1.9 V, about 2.0 V, about 2.1 V, about 2.2 V,about 2.3 V, about 2.4 V, about 2.5 V, about 2.6 V, about 2.7 V, 2.8 V,about 2.9 V, about 3.0 V, about 3.1 V, about 3.2 V, about 3.3 V, about3.4 V, about 3.5 V, about 3.6 V, about 3.7 V, about 3.8 V, about 3.9 V,about 4.0 V, about 4.1 V, about 4.2 V, about 4.3 V, 4.4 V, 4.5 V, about4.6 V, about 4.7 V, 4.8 V, about 4.9 V, about 5.0 V, about 5.1 V, about5.2 V, about 5.3 V, about 5.4 V, about 5.5 V, about 5.6 V, about 5.7 V,about 5.8 V, about 5.9 V, about 6.0 V, or the like.

In embodiments, LLEC systems disclosed herein can produce a low levelelectric current of between for example about 1 and about 200micro-amperes, between about 10 and about 190 micro-amperes, betweenabout 20 and about 180 micro-amperes, between about 30 and about 170micro-amperes, between about 40 and about 160 micro-amperes, betweenabout 50 and about 150 micro-amperes, between about 60 and about 140micro-amperes, between about 70 and about 130 micro-amperes, betweenabout 80 and about 120 micro-amperes, between about 90 and about 100micro-amperes, or the like.

In an embodiment, an LLEC system disclosed herein can produce a lowlevel electric current of between for example about 1 and about 10micro-amperes

In embodiments, LLEC systems disclosed herein can produce a low levelmicro-current of between for example about 1 and about 400micro-amperes, between about 20 and about 380 micro-amperes, betweenabout 400 and about 360 micro-amperes, between about 60 and about 340micro-amperes, between about 80 and about 320 micro-amperes, betweenabout 100 and about 3000 micro-amperes, between about 120 and about 280micro-amperes, between about 140 and about 260 micro-amperes, betweenabout 160 and about 240 micro-amperes, between about 180 and about 220micro-amperes, or the like.

In embodiments, LLEC systems disclosed herein can produce a low levelmicro-current about 10 micro-amperes, about 20 micro-amperes, about 30micro-amperes, about 40 micro-amperes, about 50 micro-amperes, about 60micro-amperes, about 70 micro-amperes, about 80 micro-amperes, about 90micro-amperes, about 100 micro-amperes, about 110 micro-amperes, about120 micro-amperes, about 130 micro-amperes, about 140 micro-amperes,about 150 micro-amperes, about 160 micro-amperes, about 170micro-amperes, about 180 micro-amperes, about 190 micro-amperes, about200 micro-amperes, about 210 micro-amperes, about 220 micro-amperes,about 240 micro-amperes, about 260 micro-amperes, about 280micro-amperes, about 300 micro-amperes, about 320 micro-amperes, about340 micro-amperes, about 360 micro-amperes, about 380 micro-amperes,about 400 micro-amperes, or the like.

In embodiments, the disclosed LLEC systems can produce a low levelmicro-current of not more than 10 micro-amperes, or not more than about20 micro-amperes, not more than about 30 micro-amperes, not more thanabout 40 micro-amperes, not more than about 50 micro-amperes, not morethan about 60 micro-amperes, not more than about 70 micro-amperes, notmore than about 80 micro-amperes, not more than about 90 micro-amperes,not more than about 100 micro-amperes, not more than about 110micro-amperes, not more than about 120 micro-amperes, not more thanabout 130 micro-amperes, not more than about 140 micro-amperes, not morethan about 150 micro-amperes, not more than about 160 micro-amperes, notmore than about 170 micro-amperes, not more than about 180micro-amperes, not more than about 190 micro-amperes, not more thanabout 200 micro-amperes, not more than about 210 micro-amperes, not morethan about 220 micro-amperes, not more than about 230 micro-amperes, notmore than about 240 micro-amperes, not more than about 250micro-amperes, not more than about 260 micro-amperes, not more thanabout 270 micro-amperes, not more than about 280 micro-amperes, not morethan about 290 micro-amperes, not more than about 300 micro-amperes, notmore than about 310 micro-amperes, not more than about 320micro-amperes, not more than about 340 micro-amperes, not more thanabout 360 micro-amperes, not more than about 380 micro-amperes, not morethan about 400 micro-amperes, not more than about 420 micro-amperes, notmore than about 440 micro-amperes, not more than about 460micro-amperes, not more than about 480 micro-amperes, or the like.

In embodiments, LLEC systems disclosed herein can produce a low levelmicro-current of not less than 10 micro-amperes, not less than 20micro-amperes, not less than 30 micro-amperes, not less than 40micro-amperes, not less than 50 micro-amperes, not less than 60micro-amperes, not less than 70 micro-amperes, not less than 80micro-amperes, not less than 90 micro-amperes, not less than 100micro-amperes, not less than 110 micro-amperes, not less than 120micro-amperes, not less than 130 micro-amperes, not less than 140micro-amperes, not less than 150 micro-amperes, not less than 160micro-amperes, not less than 170 micro-amperes, not less than 180micro-amperes, not less than 190 micro-amperes, not less than 200micro-amperes, not less than 210 micro-amperes, not less than 220micro-amperes, not less than 230 micro-amperes, not less than 240micro-amperes, not less than 250 micro-amperes, not less than 260micro-amperes, not less than 270 micro-amperes, not less than 280micro-amperes, not less than 290 micro-amperes, not less than 300micro-amperes, not less than 310 micro-amperes, not less than 320micro-amperes, not less than 330 micro-amperes, not less than 340micro-amperes, not less than 350 micro-amperes, not less than 360micro-amperes, not less than 370 micro-amperes, not less than 380micro-amperes, not less than 390 micro-amperes, not less than 400micro-amperes, or the like.

The applied electrodes or reservoirs or dots can adhere or bond to theprimary surface 2 because a biocompatible binder is mixed, inembodiments into separate mixtures, with each of the dissimilar metalsthat will create the pattern of voltaic cells, in embodiments. Most inksare simply a carrier, and a binder mixed with pigment. Similarly,conductive metal solutions can be a binder mixed with a conductiveelement. The resulting conductive metal solutions can be used with anapplication method such as screen printing to apply the electrodes tothe primary surface in predetermined patterns. Once the conductive metalsolutions dry and/or cure, the patterns of spaced electrodes cansubstantially maintain their relative position, even on a flexiblematerial such as that used for an LLEC or LLEF system. To make a limitednumber of the systems of an embodiment disclosed herein, the conductivemetal solutions can be hand applied onto a common adhesive bandage sothat there is an array of alternating electrodes that are spaced about amillimeter apart on the primary surface of the bandage. The solution canbe allowed to dry before being applied to a surface so that theconductive materials do not mix, which could interrupt the array andcause direct reactions that will release the elements.

In certain embodiments that utilize a poly-cellulose binder, the binderitself can have an beneficial effect such as reducing the localconcentration of matrix metallo-proteases through an iontophoreticprocess that drives the cellulose into the surrounding tissue. Thisprocess can be used to electronically drive other components such asdrugs into the surrounding tissue.

The binder can include any biocompatible liquid material that can bemixed with a conductive element (preferably metallic crystals of silveror zinc) to create a conductive solution which can be applied as a thincoating to a surface. One suitable binder is a solvent reduciblepolymer, such as the polyacrylic non-toxic silk-screen ink manufacturedby COLORCON® Inc., a division of Berwind Pharmaceutical Services, Inc.(see COLORCON® NO-TOX® product line, part number NT28). In an embodimentthe binder is mixed with high purity (at least 99.999%) metallic silvercrystals to make the silver conductive solution. The silver crystals,which can be made by grinding silver into a powder, are preferablysmaller than 100 microns in size or about as fine as flour. In anembodiment, the size of the crystals is about 325 mesh, which istypically about 40 microns in size or a little smaller. The binder isseparately mixed with high purity (at least 99.99%, in an embodiment)metallic zinc powder which has also preferably been sifted throughstandard 325 mesh screen, to make the zinc conductive solution. Forbetter quality control and more consistent results, most of the crystalsused should be larger than 325 mesh and smaller than 200 mesh. Forexample the crystals used should be between 200 mesh and 325 mesh, orbetween 210 mesh and 310 mesh, between 220 mesh and 300 mesh, between230 mesh and 290 mesh, between 240 mesh and 280 mesh, between 250 meshand 270 mesh, between 255 mesh and 265 mesh, or the like.

Other powders of metal can be used to make other conductive metalsolutions in the same way as described in other embodiments.

The size of the metal crystals, the availability of the surface to theconductive fluid and the ratio of metal to binder affects the releaserate of the metal from the mixture. When COLORCON® polyacrylic ink isused as the binder, about 10 to 40 percent of the mixture should bemetal for a longer term bandage (for example, one that stays on forabout 10 days). For example, for a longer term LLEC or LLEF system thepercent of the mixture that should be metal can be 8 percent, or 10percent, 12 percent, 14 percent, 16 percent, 18 percent, 20 percent, 22percent, 24 percent, 26 percent, 28 percent, 30 percent, 32 percent, 34percent, 36 percent, 38 percent, 40 percent, 42 percent, 44 percent, 46percent, 48 percent, 50 percent, or the like.

If the same binder is used, but the percentage of the mixture that ismetal is increased to 60 percent or higher, then the release rate willbe much faster and a typical system will only be effective for a fewdays. For example, for a shorter term device, the percent of the mixturethat should be metal can be 40 percent, or 42 percent, 44 percent, 46percent, 48 percent, 50 percent, 52 percent, 54 percent, 56 percent, 58percent, 60 percent, 62 percent, 64 percent, 66 percent, 68 percent, 70percent, 72 percent, 74 percent, 76 percent, 78 percent, 80 percent, 82percent, 84 percent, 86 percent, 88 percent, 90 percent, or the like.

For LLEC or LLEF systems comprising a pliable substrate it can bedesirable to decrease the percentage of metal down to, for example, 5percent or less, or to use a binder that causes the crystals to be moredeeply embedded, so that the primary surface will be antimicrobial for avery long period of time and will not wear prematurely. Other binderscan dissolve or otherwise break down faster or slower than a polyacrylicink, so adjustments can be made to achieve the desired rate ofspontaneous reactions from the voltaic cells.

To maximize the number of voltaic cells, in various embodiments, apattern of alternating silver masses or electrodes or reservoirs andzinc masses or electrodes or reservoirs can create an array ofelectrical currents across the primary surface. A basic pattern, shownin FIG. 1, has each mass of silver equally spaced from four masses ofzinc, and has each mass of zinc equally spaced from four masses ofsilver, according to an embodiment. The first electrode 6 is separatedfrom the second electrode 10 by a spacing 8. The designs of firstelectrode 6 and second electrode 10 are simply round dots, and in anembodiment, are repeated. Numerous repetitions 12 of the designs resultin a pattern. For an exemplary device comprising silver and zinc, eachsilver design preferably has about twice as much mass as each zincdesign, in an embodiment. For the pattern in FIG. 1, the silver designsare most preferably about a millimeter from each of the closest fourzinc designs, and vice-versa. The resulting pattern of dissimilar metalmasses defines an array of voltaic cells when introduced to anelectrolytic solution. Further disclosure relating to methods ofproducing micro-arrays can be found in U.S. Pat. No. 7,813,806 entitledCURRENT PRODUCING SURFACE FOR TREATING BIOLOGIC TISSUE issued Oct. 12,2010, which is incorporated by reference in its entirety.

A dot pattern of masses like the alternating round dots of FIG. 1 can bepreferred when applying conductive material onto a flexible material,such as those used for a surgical or medical mask or respirator, as thedots won't significantly affect the flexibility of the material. Tomaximize the density of electrical current over a primary surface thepattern of FIG. 2 can be used. The first electrode 6 in FIG. 2 is alarge hexagonally shaped dot, and the second electrode 10 is a pair ofsmaller hexagonally shaped dots that are spaced from each other. Thespacing 8 that is between the first electrode 6 and the second electrode10 maintains a relatively consistent distance between adjacent sides ofthe designs. Numerous repetitions 12 of the designs result in a pattern14 that can be described as at least one of the first design beingsurrounded by six hexagonally shaped dots of the second design. Inembodiments, electrodes can be deposited or placed on both sides of aplanar substrate, for example, both sides of a mask, for example bothsides of (interior facing the patient and exterior facing away from thepatient) a neck gaiter.

FIGS. 3 and 4 show how the pattern of FIG. 2 can be used to make anembodiment disclosed herein. The pattern shown in detail in FIG. 2 isapplied to the primary surface 2 of an embodiment. The back 20 of theprinted material is fixed to a substrate layer 22. This layer isadhesively fixed to a pliable layer 16.

FIG. 5 shows an additional feature, which can be added between designs,that can initiate the flow of current in a poor electrolytic solution. Afine line 24 is printed using one of the conductive metal solutionsalong a current path of each voltaic cell. The fine line will initiallyhave a direct reaction but will be depleted until the distance betweenthe electrodes increases to where maximum voltage is realized. Theinitial current produced is intended to help control edema so that theLLEC system will be effective. If the electrolytic solution is highlyconductive when the system is initially applied the fine line can bequickly depleted and the device will function as though the fine linehad never existed.

FIGS. 6 and 7 show alternative patterns that use at least one linedesign. The first electrode 6 of FIG. 6 is a round dot similar to thefirst design used in FIG. 1. The second electrode 10 of FIG. 6 is aline. When the designs are repeated, they define a pattern of parallellines that are separated by numerous spaced dots. FIG. 7 uses only linedesigns. The first electrode 6 can be thicker or wider than the secondelectrode 10 if the oxidation-reduction reaction requires more metalfrom the first conductive element (mixed into the first design'sconductive metal solution) than the second conductive element (mixedinto the second design's conductive metal solution). The lines can bedashed. Another pattern can be silver grid lines that have zinc massesin the center of each of the cells of the grid. The pattern can beletters printed from alternating conductive materials so that a messagecan be printed onto the primary surface such as a brand name oridentifying information such as patient blood type.

Because the spontaneous oxidation-reduction reaction of silver and zincuses a ratio of approximately two silver to one zinc, the silver designcan contain about twice as much mass as the zinc design in anembodiment. At a spacing of about 1 mm between the closest dissimilarmetals (closest edge to closest edge) each voltaic cell that contacts aconductive fluid can create approximately 1 volt of potential that willpenetrate substantially through the dermis and epidermis. Closer spacingof the dots can decrease the resistance, providing less potential, andthe current will not penetrate as deeply. If the spacing falls belowabout one tenth of a millimeter a benefit of the spontaneous reaction isthat which is also present with a direct reaction; silver can beelectrically driven into the skin. Therefore, spacing between theclosest conductive materials can be 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2 mm, 2.1 mm, 2.2 mm, 2.3mm, 2.4 mm, 2.5 mm, 2.6 mm, 2.7 mm, 2.8 mm, 2.9 mm, 3 mm, 3.1 mm, 3.2mm, 3.3 mm, 3.4 mm, 3.5 mm, 3.6 mm, 3.7 mm, 3.8 mm, 3.9 mm, 4 mm, 4.1mm, 4.2 mm, 4.3 mm, 4.4 mm, 4.5 mm, 4.6 mm, 4.7 mm, 4.8 mm, 4.9 mm, 5mm, 5.1 mm, 5.2 mm, 5.3 mm, 5.4 mm, 5.5 mm, 5.6 mm, 5.7 mm, 5.8 mm, 5.9mm, 6 mm, or the like.

In certain embodiments the spacing between the closest conductivematerials can be not more than 0.1 mm, not more than 0.2 mm, not morethan 0.3 mm, not more than 0.4 mm, not more than 0.5 mm, not more than0.6 mm, not more than 0.7 mm, not more than 0.8 mm, not more than 0.9mm, not more than 1 mm, not more than 1.1 mm, not more than 1.2 mm, notmore than 1.3 mm, not more than 1.4 mm, not more than 1.5 mm, not morethan 1.6 mm, not more than 1.7 mm, not more than 1.8 mm, not more than1.9 mm, not more than 2 mm, not more than 2.1 mm, not more than 2.2 mm,not more than 2.3 mm, not more than 2.4 mm, not more than 2.5 mm, notmore than 2.6 mm, not more than 2.7 mm, not more than 2.8 mm, not morethan 2.9 mm, not more than 3 mm, not more than 3.1 mm, not more than 3.2mm, not more than 3.3 mm, not more than 3.4 mm, not more than 3.5 mm,not more than 3.6 mm, not more than 3.7 mm, not more than 3.8 mm, notmore than 3.9 mm, not more than 4 mm, not more than 4.1 mm, not morethan 4.2 mm, not more than 4.3 mm, not more than 4.4 mm, not more than4.5 mm, not more than 4.6 mm, not more than 4.7 mm, not more than 4.8mm, not more than 4.9 mm, not more than 5 mm, not more than 5.1 mm, notmore than 5.2 mm, not more than 5.3 mm, not more than 5.4 mm, not morethan 5.5 mm, not more than 5.6 mm, not more than 5.7 mm, not more than5.8 mm, not more than 5.9 mm, not more than 6 mm, or the like.

In certain embodiments spacing between the closest conductive materialscan be not less than 0.1 mm, not less than 0.2 mm, not less than 0.3 mm,not less than 0.4 mm, not less than 0.5 mm, not less than 0.6 mm, notless than 0.7 mm, not less than 0.8 mm, not less than 0.9 mm, not lessthan 1 mm, not less than 1.1 mm, not less than 1.2 mm, not less than 1.3mm, not less than 1.4 mm, not less than 1.5 mm, not less than 1.6 mm,not less than 1.7 mm, not less than 1.8 mm, not less than 1.9 mm, notless than 2 mm, not less than 2.1 mm, not less than 2.2 mm, not lessthan 2.3 mm, not less than 2.4 mm, not less than 2.5 mm, not less than2.6 mm, not less than 2.7 mm, not less than 2.8 mm, not less than 2.9mm, not less than 3 mm, not less than 3.1 mm, not less than 3.2 mm, notless than 3.3 mm, not less than 3.4 mm, not less than 3.5 mm, not lessthan 3.6 mm, not less than 3.7 mm, not less than 3.8 mm, not less than3.9 mm, not less than 4 mm, not less than 4.1 mm, not less than 4.2 mm,not less than 4.3 mm, not less than 4.4 mm, not less than 4.5 mm, notless than 4.6 mm, not less than 4.7 mm, not less than 4.8 mm, not lessthan 4.9 mm, not less than 5 mm, not less than 5.1 mm, not less than 5.2mm, not less than 5.3 mm, not less than 5.4 mm, not less than 5.5 mm,not less than 5.6 mm, not less than 5.7 mm, not less than 5.8 mm, notless than 5.9 mm, not less than 6 mm, or the like.

Disclosed herein include LLEC or LLEF systems comprising a primarysurface of a pliable material wherein the pliable material is adapted tobe applied to an area of tissue such as the face and neck of a subject;a first electrode design formed from a first conductive liquid thatincludes a mixture of a polymer and a first element, the firstconductive liquid being applied into a position of contact with theprimary surface, the first element including a metal species, and thefirst electrode design including at least one dot or reservoir, whereinselective ones of the at least one dot or reservoir have approximately a1.5 mm +/− 1 mm mean diameter; a second electrode design formed from asecond conductive liquid that includes a mixture of a polymer and asecond element, the second element including a different metal speciesthan the first element, the second conductive liquid being printed intoa position of contact with the primary surface, and the second electrodedesign including at least one other dot or reservoir, wherein selectiveones of the at least one other dot or reservoir have approximately a 2.5mm +/− 2 mm mean diameter; a spacing on the primary surface that isbetween the first electrode design and the second electrode design suchthat the first electrode design does not physically contact the secondelectrode design, wherein the spacing is approximately 1.5 mm +/− 1 mm,and at least one repetition of the first electrode design and the secondelectrode design, the at least one repetition of the first electrodedesign being substantially adjacent the second electrode design, whereinthe at least one repetition of the first electrode design and the secondelectrode design, in conjunction with the spacing between the firstelectrode design and the second electrode design, defines at least onepattern of at least one voltaic cell for spontaneously generating atleast one electrical current when introduced to an electrolyticsolution. Therefore, in embodiments, electrodes, dots or reservoirs canhave a mean diameter of 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm,0.8 mm, 0.9 mm, 1.0 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm,1.7 mm, 1.8 mm, 1.9 mm, 2.0 mm, 2.1 mm, 2.2 mm, 2.3 mm, 2.4 mm, 2.5 mm,2.6 mm, 2.7 mm, 2.8 mm, 2.9 mm, 3.0 mm, 3.1 mm, 3.2 mm, 3.3 mm, 3.4 mm,3.5 mm, 3.6 mm, 3.7 mm, 3.8 mm, 3.9 mm, 4.0 mm, 4.1 mm, 4.2 mm, 4.3 mm,4.4 mm, 4.5 mm, 4.6 mm, 4.7 mm, 4.8 mm, 4.9 mm, 5.0 mm, or the like.

In further embodiments, electrodes, dots or reservoirs can have a meandiameter of not less than 0.2 mm, not less than 0.3 mm, not less than0.4 mm, not less than 0.5 mm, not less than 0.6 mm, not less than 0.7mm, not less than 0.8 mm, not less than 0.9 mm, not less than 1.0 mm,not less than 1.1 mm, not less than 1.2 mm, not less than 1.3 mm, notless than 1.4 mm, not less than 1.5 mm, not less than 1.6 mm, not lessthan 1.7 mm, not less than 1.8 mm, not less than 1.9 mm, not less than2.0 mm, not less than 2.1 mm, not less than 2.2 mm, not less than 2.3mm, not less than 2.4 mm, not less than 2.5 mm, not less than 2.6 mm,not less than 2.7 mm, not less than 2.8 mm, not less than 2.9 mm, notless than 3.0 mm, not less than 3.1 mm, not less than 3.2 mm, not lessthan 3.3 mm, not less than 3.4 mm, not less than 3.5 mm, not less than3.6 mm, not less than 3.7 mm, not less than 3.8 mm, not less than 3.9mm, not less than 4.0 mm, not less than 4.1 mm, not less than 4.2 mm,not less than 4.3 mm, not less than 4.4 mm, not less than 4.5 mm, notless than 4.6 mm, not less than 4.7 mm, not less than 4.8 mm, not lessthan 4.9 mm, not less than 5.0 mm, or the like.

In further embodiments, electrodes, dots or reservoirs can have a meandiameter of not more than 0.2 mm, not more than 0.3 mm, not more than0.4 mm, not more than 0.5 mm, not more than 0.6 mm, not more than 0.7mm, not more than 0.8 mm, not more than 0.9 mm, not more than 1.0 mm,not more than 1.1 mm, not more than 1.2 mm, not more than 1.3 mm, notmore than 1.4 mm, not more than 1.5 mm, not more than 1.6 mm, not morethan 1.7 mm, not more than 1.8 mm, not more than 1.9 mm, not more than2.0 mm, not more than 2.1 mm, not more than 2.2 mm, not more than 2.3mm, not more than 2.4 mm, not more than 2.5 mm, not more than 2.6 mm,not more than 2.7 mm, not more than 2.8 mm, not more than 2.9 mm, notmore than 3.0 mm, not more than 3.1 mm, not more than 3.2 mm, not morethan 3.3 mm, not more than 3.4 mm, not more than 3.5 mm, not more than3.6 mm, not more than 3.7 mm, not more than 3.8 mm, not more than 3.9mm, not more than 4.0 mm, not more than 4.1 mm, not more than 4.2 mm,not more than 4.3 mm, not more than 4.4 mm, not more than 4.5 mm, notmore than 4.6 mm, not more than 4.7 mm, not more than 4.8 mm, not morethan 4.9 mm, not more than 5.0 mm, or the like.

In embodiments, the density of the conductive materials can be, forexample, 20 reservoirs per square inch (/in²), 30 reservoirs /in², 40reservoirs /in², 50 reservoirs /in², 60 reservoirs /in², 70 reservoirs/in², 80 reservoirs /in², r 90 reservoirs /in², 100 reservoirs /in², 150reservoirs /in², 200 reservoirs /in², 250 reservoirs /in², 300reservoirs /in², or 350 reservoirs /in², 400 reservoirs /in², 450reservoirs /in², 500 reservoirs /in², 550 reservoirs /in², 600reservoirs /in², 650 reservoirs /in², 700 reservoirs /in², 750reservoirs /in², more, or the like.

In embodiments, the density of the conductive materials can be, forexample, more than 20 reservoirs /in², more than 30 reservoirs /in²,more than 40 reservoirs /in², more than 50 reservoirs /in², more than 60reservoirs /in², more than 70 reservoirs /in², more than 80 reservoirs/in², more than 90 reservoirs /in², more than 100 reservoirs /in², morethan 150 reservoirs /in², more than 200 reservoirs /in², more than 250reservoirs /in², more than 300 reservoirs /in², more than 350 reservoirs/in², more than 400 reservoirs /in², more than 450 reservoirs /in², morethan 500 reservoirs /in², more than 550 reservoirs /in², more than 600reservoirs /in², more than 650 reservoirs /in², more than 700 reservoirs/in², more than 750 reservoirs /in², or more, or the like.

The material concentrations or quantities within and/or the relativesizes (e.g., dimensions or surface area) of the first and secondreservoirs can be selected deliberately to achieve variouscharacteristics of the systems' behavior. For example, the quantities ofmaterial within a first and second reservoir can be selected to providean apparatus having an operational behavior that depletes atapproximately a desired rate and/or that “dies” after an approximateperiod of time after activation. In an embodiment the one or more firstreservoirs and the one or more second reservoirs are configured tosustain one or more currents for an approximate pre-determined period oftime, after activation. It is to be understood that the amount of timethat currents are sustained can depend on external conditions andfactors (e.g., the quantity and type of activation material), andcurrents can occur intermittently depending on the presence or absenceof activation material. Further disclosure relating to producingreservoirs that are configured to sustain one or more currents for anapproximate pre-determined period of time can be found in U.S. Pat. No.7,904,147 entitled SUBSTANTIALLY PLANAR ARTICLE AND METHODS OFMANUFACTURE issued Mar. 8, 2011, which is incorporated by referenceherein in its entirety.

In various embodiments the difference of the standard potentials of thefirst and second reservoirs can be in a range from 0.05 V toapproximately 5.0 V. For example, the standard potential can be 0.05 V,or 0.06 V, 0.07 V, 0.08 V, 0.09 V, 0.1 V, 0.2 V, 0.3 V, 0.4 V, 0.5 V,0.6 V, 0.7 V, 0.8 V, 0.9 V, 1.0 V, 1.1 V, 1.2 V, 1.3 V, 1.4 V, 1.5 V,1.6 V, 1.7 V, 1.8 V, 1.9 V, 2.0 V, 2.1 V, 2.2 V, 2.3 V, 2.4 V, 2.5 V,2.6 V, 2.7 V, 2.8 V, 2.9 V, 3.0 V, 3.1 V, 3.2 V, 3.3 V, 3.4 V, 3.5 V,3.6 V, 3.7 V, 3.8 V, 3.9 V, 4.0 V, 4.1 V, 4.2 V, 4.3 V, 4.4 V, 4.5 V,4.6 V, 4.7 V, 4.8 V, 4.9 V, 5.0 V, or the like.

In a particular embodiment the difference of the standard potentials ofthe first and second reservoirs can be at least 0.05 V, at least 0.06 V,at least 0.07 V, at least 0.08 V, at least 0.09 V, at least 0.1 V, atleast 0.2 V, at least 0.3 V, at least 0.4 V, at least 0.5 V, at least0.6 V, at least 0.7 V, at least 0.8 V, at least 0.9 V, at least 1.0 V,at least 1.1 V, at least 1.2 V, at least 1.3 V, at least 1.4 V, at least1.5 V, at least 1.6 V, at least 1.7 V, at least 1.8 V, at least 1.9 V,at least 2.0 V, at least 2.1 V, at least 2.2 V, at least 2.3 V, at least2.4 V, at least 2.5 V, at least 2.6 V, at least 2.7 V, at least 2.8 V,at least 2.9 V, at least 3.0 V, at least 3.1 V, at least 3.2 V, at least3.3 V, at least 3.4 V, at least 3.5 V, at least 3.6 V, at least 3.7 V,at least 3.8 V, at least 3.9 V, at least 4.0 V, at least 4.1 V, at least4.2 V, at least 4.3 V, at least 4.4 V, at least 4.5 V, at least 4.6 V,at least 4.7 V, at least 4.8 V, at least 4.9 V, at least 5.0 V, or thelike.

In a particular embodiment, the difference of the standard potentials ofthe first and second reservoirs can be not more than 0.05 V, not morethan 0.06 V, not more than 0.07 V, not more than 0.08 V, not more than0.09 V, not more than 0.1 V, not more than 0.2 V, not more than 0.3 V,not more than 0.4 V, not more than 0.5 V, not more than 0.6 V, not morethan 0.7 V, not more than 0.8 V, not more than 0.9 V, not more than 1.0V, not more than 1.1 V, not more than 1.2 V, not more than 1.3 V, notmore than 1.4 V, not more than 1.5 V, not more than 1.6 V, not more than1.7 V, not more than 1.8 V, not more than 1.9 V, not more than 2.0 V,not more than 2.1 V, not more than 2.2 V, not more than 2.3 V, not morethan 2.4 V, not more than 2.5 V, not more than 2.6 V, not more than 2.7V, not more than 2.8 V, not more than 2.9 V, not more than 3.0 V, notmore than 3.1 V, not more than 3.2 V, not more than 3.3 V, not more than3.4 V, not more than 3.5 V, not more than 3.6 V, not more than 3.7 V,not more than 3.8 V, not more than 3.9 V, not more than 4.0 V, not morethan 4.1 V, not more than 4.2 V, not more than 4.3 V, not more than 4.4V, not more than 4.5 V, not more than 4.6 V, not more than 4.7 V, notmore than 4.8 V, not more than 4.9 V, not more than 5.0 V, or the like.In embodiments that include very small reservoirs (e.g., on thenanometer scale), the difference of the standard potentials can besubstantially less or more. The electrons that pass between the firstreservoir and the second reservoir can be generated as a result of thedifference of the standard potentials. Further disclosure relating tostandard potentials can be found in U.S. Pat. No. 8,224,439 entitledBATTERIES AND METHODS OF MANUFACTURE AND USE issued Jul. 17, 2012, whichis incorporated be reference herein in its entirety.

The voltage present at the site of treatment is typically in the rangeof millivolts but disclosed embodiments can introduce a much highervoltage, for example near 1 volt when using the 1 mm spacing ofdissimilar metals already described. The higher voltage is believed todrive the current deeper into the treatment area. In this way thecurrent not only can drive silver and zinc into the treatment if desiredfor treatment, but the current can also provide a stimulatory current sothat the entire surface area can be treated. The higher voltage may alsoincrease antimicrobial effect bacteria and preventing biofilms. Theelectric field can also have beneficial effects on cell migration, ATPproduction, and angiogenesis.

While various embodiments have been shown and described, it will berealized that alterations and modifications can be made thereto withoutdeparting from the scope of the following claims. It is expected thatother methods of applying the conductive material can be substituted asappropriate. Also, there are numerous shapes, sizes and patterns ofvoltaic cells that have not been described but it is expected that thisdisclosure will enable those skilled in the art to incorporate their owndesigns which will then be applied to a surface to create voltaic cellswhich will become active when brought into contact with an electrolyticsolution.

Certain embodiments include LLEC or LLEF systems comprising embodimentsdesigned to be used on irregular, non-planar, or “stretching” surfaces.Embodiments disclosed herein can be used with numerous irregularsurfaces of the body, including the face, neck, etc.

In certain embodiments, the substrate can be shaped to fit a particularregion of the body. As shown in FIG. 9, an eye mask-shaped substrate canbe used for the treatment around the face and forehead. FIGS. 23-26 and34 show additional embodiments for use in covering a user's mouth andnose. Covers and inserts as disclosed herein can be attached todisclosed neck gaiters, for example reversibly attached such that aftera period of time, the insert or cover can be removed and a new insert orcover employed. In embodiments, the removed insert or cover can bewashed and reused.

Embodiments can also include means for securing the neck gaiter to theuser's head. In an embodiment the portion of the mask or substrate thatis to contact the skin comprises a multi-array matrix of biocompatiblemicrocells. In certain embodiments a fluid or cream such as a conductivefluid or cream can be applied between the multi-array matrix ofbiocompatible microcells and the skin.

Embodiments can comprise a moisture-sensitive component that changescolor when the device is activated and producing an electric current.Embodiments can comprise a moisture-sensitive component that indicateswhen the device is activated and producing an electric current. Forexample, disclosed embodiments comprise an indicator changes color aslong as the device is activated and producing an electric current.

Various apparatus embodiments which can be referred to as “medicalbatteries” are described herein. Further disclosure relating to thistechnology can be found in U.S. Pat. No. 7,672,719 entitled BATTERIESAND METHODS OF MANUFACTURE AND USE issued Mar. 2, 2010, which isincorporated herein by reference in its entirety.

Certain embodiments disclosed herein include a method of manufacturingan LLEC or LLEF system, the method comprising joining with a substratemultiple first reservoirs wherein selected ones of the multiple firstreservoirs include a reducing agent, and wherein first reservoirsurfaces of selected ones of the multiple first reservoirs are proximateto a first substrate surface; and joining with the substrate multiplesecond reservoirs wherein selected ones of the multiple secondreservoirs include an oxidizing agent, and wherein second reservoirsurfaces of selected ones of the multiple second reservoirs areproximate to the first substrate surface, wherein joining the multiplefirst reservoirs and joining the multiple second reservoirs comprisesjoining using tattooing. In embodiments the substrate can comprisegauzes comprising dots or electrodes.

Further embodiments can include a method of manufacturing an LLEC orLLEF system, the method comprising joining with a substrate multiplefirst reservoirs wherein selected ones of the multiple first reservoirsinclude a reducing agent, and wherein first reservoir surfaces ofselected ones of the multiple first reservoirs are proximate to a firstsubstrate surface; and joining with the substrate multiple secondreservoirs wherein selected ones of the multiple second reservoirsinclude an oxidizing agent, and wherein second reservoir surfaces ofselected ones of the multiple second reservoirs are proximate to thefirst substrate surface, wherein joining the multiple first reservoirsand joining the multiple second reservoirs comprises: combining themultiple first reservoirs, the multiple second reservoirs, and multipleparallel insulators to produce a pattern repeat arranged in a firstdirection across a plane, the pattern repeat including a sequence of afirst one of the parallel insulators, one of the multiple firstreservoirs, a second one of the parallel insulators, and one of themultiple second reservoirs; and weaving multiple transverse insulatorsthrough the first parallel insulator, the one first reservoir, thesecond parallel insulator, and the one second reservoir in a seconddirection across the plane to produce a woven apparatus.

Embodiments disclosed herein include LLEC and LLEF systems that canproduce an electrical stimulus and/or can electromotivate,electroconduct, electroinduct, electrotransport, and/or electrophoreseone or more therapeutic materials in areas of target tissue (e.g.,iontophoresis), and/or can cause one or more biologic or other materialsin proximity to, on or within target tissue to be rejuvenated. Furtherdisclosure relating to materials that can produce an electrical stimuluscan be found in U.S. Pat. No. 7,662,176 entitled FOOTWEAR APPARATUS ANDMETHODS OF MANUFACTURE AND USE issued Feb. 16, 2010, which isincorporated herein by reference in its entirety.

Embodiments disclosed herein include a multilayer fabric, for example alayer that can produce an LLEC/LLEF as described herein, a hydrationlayer, and a waterproof layer. Further embodiments comprise a layer of amultilayer neck gaiter.

Embodiments can comprise a conductive material, for example a salt, toassist in maintaining a conductive environment.

LLEC/LLEF Systems and Devices; Methods of Use

In embodiments, methods and devices disclosed herein can be used for toprevent viral transmission. For example, disclosed embodiments compriseneck gaiters, neck gaiter inserts, and the like, comprising electrodes.In embodiments the devices are washable. In embodiments the devices arereusable.

Disclosed embodiments can be used for preventing virus transmission, forexample those that can spread via direct contact, aerosol, or oralpathways.

Disclosed embodiments can be used to prevent transmission of, forexample, dsDNA viruses, ssDNA viruses, dsRNA viruses, (+)ssRNA viruses,(−)ssRNA viruses, ssRNA-RT viruses, dsDNA-RT viruses, and the like.

Disclosed embodiments can be used to prevent transmission of, forexample, Chikungunya, Cholera, Crimean-Congo haemorrhagic fever, Ebolavirus disease, Hendra virus infection, Influenza (pandemic, seasonal,zoonotic), Lassa fever, Marburg virus disease, Meningitis, MERS-CoV,Monkeypox, Nipah virus infection, Novel coronavirus (2019-nCoV), Plague,Rift Valley fever, SARS, Smallpox, Tularaemia, Yellow fever, Zika virusdisease, and the like.

In embodiments, a disclosed neck gaiter is worn by an infected mammal toprevent viral propagation.

In embodiments, a disclosed neck gaiter is worn by a healthy mammal toprevent viral acquisition.

In embodiments, a disclosed neck gaiter insert is worn by an infectedmammal to prevent viral propagation.

In embodiments, a disclosed neck gaiter insert is worn by a healthymammal to prevent viral acquisition.

In an exemplary embodiment, a method disclosed herein comprises applyinga disclosed device to an area where treatment is desired, for example,over the mouth, nose, eyes, etc.

In another exemplary embodiment, a method disclosed herein comprisesapplying a disclosed neck gaiter insert to a neck gaiter to provideincreased anti-viral performance as compared to the antiviralperformance of the neck gaiter alone.

EXAMPLES

The following non-limiting examples are provided for illustrativepurposes only in order to facilitate a more complete understanding ofrepresentative embodiments. These examples should not be construed tolimit any of the embodiments described in the present specification.

Example 1 Cell Migration Assay

The in vitro scratch assay is an easy, low-cost and well-developedmethod to measure cell migration in vitro. The basic steps involvecreating a “scratch” in a cell monolayer, capturing images at thebeginning and at regular intervals during cell migration to close thescratch, and comparing the images to quantify the migration rate of thecells. Compared to other methods, the in vitro scratch assay isparticularly suitable for studies on the effects of cell-matrix andcell-cell interactions on cell migration, mimic cell migration duringwound healing in vivo and are compatible with imaging of live cellsduring migration to monitor intracellular events if desired. In additionto monitoring migration of homogenous cell populations, this method hasalso been adopted to measure migration of individual cells in theleading edge of the scratch. Not taking into account the time fortransfection of cells, in vitro scratch assay per se usually takes fromseveral hours to overnight.

Human keratinocytes were plated under plated under placebo or an LLECsystem (labeled “PROCELLERA®”). Cells were also plated under silver-onlyor zinc-only dressings. After 24 hours, the scratch assay was performed.Cells plated under the PROCELLERA® device displayed increased migrationinto the “scratched” area as compared to any of the zinc, silver, orplacebo dressings. After 9 hours, the cells plated under the PROCELLERA®device had almost “closed” the scratch. This demonstrates the importanceof electrical activity to cell migration and infiltration.

In addition to the scratch test, genetic expression was tested.Increased insulin growth factor (IGF)-1R phosphorylation wasdemonstrated by the cells plated under the PROCELLERA®device as comparedto cells plated under insulin growth factor alone.

Integrin accumulation also affects cell migration. An increase inintegrin accumulation achieved with the LLEC system. Integrin isnecessary for cell migration, and is found on the leading edge ofmigrating cell.

Thus, the tested LLEC system enhanced cellular migration andIGF-1R/integrin involvement. This involvement demonstrates the effectthat the LLEC system had upon cell receptors involved with the woundhealing process.

Example 2 Zone of Inhibition Test

For cellular repair to be most efficient, available energy should not beshared with ubiquitous microbes. In this “zone of inhibition” test,placebo, an LLEC device (PROCELLERA®) and silver only were tested in anagar medium with a 24 hour growth of organisms. Bacterial growth waspresent over the placebo, a zone of inhibition over the PROCELLERA® anda minimal inhibition zone over the silver. Because the samples were“buried” in agar, the electricidal effect of the LLEC system could betested. Silver ion diffusion, the method used by silver basedantimicrobials, alone was not sufficient. The test demonstrates theimproved bactericidal effect of PROCELLERA® as compared to silver alone.

Example 3 LLEC Influence on Human Keratinocyte Migration

An LLEC-generated electrical field was mapped, leading to theobservation that LLEC generates hydrogen peroxide, known to drive redoxsignaling. LLEC-induced phosphorylation of redox-sensitive IGF-1R wasdirectly implicated in cell migration. The LLEC also increasedkeratinocyte mitochondrial membrane potential.

The LLEC was made of polyester printed with dissimilar elemental metalsas described herein. It comprises alternating circular regions of silverand zinc dots, along with a proprietary, biocompatible binder added tolock the electrodes to the surface of a flexible substrate in a patternof discrete reservoirs. When the LLEC contacts an aqueous solution, thesilver positive electrode (cathode) is reduced while the zinc negativeelectrode (anode) is oxidized. The LLEC used herein consisted of metalsplaced in proximity of about 1 mm to each other thus forming a redoxcouple and generating an ideal potential on the order of 1 Volt. Thecalculated values of the electric field from the LLEC were consistentwith the magnitudes that are typically applied (1-10 V/cm) in classicalelectrotaxis experiments, suggesting that cell migration observed withthe bioelectric dressing is likely due to electrotaxis.

Measurement of the potential difference between adjacent zinc and silverdots when the LLEC is in contact with de-ionized water yielded a valueof about 0.2 Volts. Though the potential difference between zinc andsilver dots can be measured, non-intrusive measurement of the electricfield arising from contact between the LLEC and liquid medium wasdifficult. Keratinocyte migration was accelerated by exposure to anAg/Zn LLEC. Replacing the Ag/Zn redox couple with Ag or Zn alone did notreproduce the effect of keratinocyte acceleration.

Exposing keratinocytes to an LLEC for 24 h significantly increased greenfluorescence in the dichlorofluorescein (DCF) assay indicatinggeneration of reactive oxygen species under the effect of the LLEC. Todetermine whether H₂O₂ is generated specifically, keratinocytes werecultured with an LLEC or placebo for 24 h and then loaded with PF6-AM(Peroxyfluor-6 acetoxymethyl ester; an indicator of endogenous H₂O₂).Greater intracellular fluorescence was observed in the LLECkeratinocytes compared to the cells grown with placebo. Over-expressionof catalase (an enzyme that breaks down H₂O₂) attenuated the increasedmigration triggered by the LLEC. Treating keratinocytes with N-AcetylCysteine (which blocks oxidant-induced signaling) also failed toreproduce the increased migration observed with LLEC. Thus, H₂O₂signaling mediated the increase of keratinocyte migration under theeffect of the electrical stimulus.

External electrical stimulus can up-regulate the TCA (tricarboxylicacid) cycle. The stimulated TCA cycle is then expected to generate moreNADH and FADH₂ to enter into the electron transport chain and elevatethe mitochondrial membrane potential (Δm). Fluorescent dyes JC-1 andTMRM were used to measure mitochondrial membrane potential. JC-1 is alipophilic dye which produces a red fluorescence with high Δm and greenfluorescence when Am is low. TMRM produces a red fluorescenceproportional to Δm. Treatment of keratinocytes with LLEC for 24 hdemonstrated significantly high red fluorescence with both JC-1 andTMRM, indicating an increase in mitochondrial membrane potential andenergized mitochondria under the effect of the LLEC. As a potentialconsequence of a stimulated TCA cycle, available pyruvate (the primarysubstrate for the TCA cycle) is depleted resulting in an enhanced rateof glycolysis. This can lead to an increase in glucose uptake in orderto push the glycolytic pathway forward. The rate of glucose uptake inHaCaT cells treated with LLEC was examined next. More than two foldenhancement of basal glucose uptake was observed after treatment withLLEC for 24 h as compared to placebo control.

Keratinocyte migration is known to involve phosphorylation of a numberof receptor tyrosine kinases (RTKs). To determine which RTKs areactivated as a result of LLEC, scratch assay was performed onkeratinocytes treated with LLEC or placebo for 24 h. Samples werecollected after 3 h and an antibody array that allows simultaneousassessment of the phosphorylation status of 42 RTKs was used to quantifyRTK phosphorylation. It was determined that LLEC significantly inducesIGF-1R phosphorylation. Sandwich ELISA using an antibody againstphospho-IGF-1R and total IGF-1R verified this determination. As observedwith the RTK array screening, potent induction in phosphorylation ofIGF-1R was observed 3 h post scratch under the influence of LLEC. IGF-1Rinhibitor attenuated the increased keratinocyte migration observed withLLEC treatment.

MBB (monobromobimane) alkylates thiol groups, displacing the bromine andadding a fluorescent tag (lamda emission=478 nm). MCB (monochlorobimane)reacts with only low molecular weight thiols such as glutathione.Fluorescence emission from UV laser-excited keratinocytes loaded witheither MBB or MCB was determined for 30 min. Mean fluorescence collectedfrom 10,000 cells showed a significant shift of MBB fluorescenceemission from cells. No significant change in MCB fluorescence wasobserved, indicating a change in total protein thiol but notglutathione. HaCaT cells were treated with LLEC for 24 h followed by ascratch assay. Integrin expression was observed by immuno-cytochemistryat different time points. Higher integrin expression was observed 6 hpost scratch at the migrating edge.

Consistent with evidence that cell migration requires H₂O₂ sensing, wedetermined that by blocking H₂O₂ signaling by decomposition of H₂O₂ bycatalase or ROS scavenger, N-acetyl cysteine, the increase in LLEC-driven cell migration is prevented. The observation that the LLECincreases H₂O₂ production is significant because in addition to cellmigration, hydrogen peroxide generated in the wound margin tissue isrequired to recruit neutrophils and other leukocytes to the wound,regulates monocyte function, and VEGF signaling pathway and tissuevascularization. Therefore, external electrical stimulation can be usedas an effective strategy to deliver low levels of hydrogen peroxide overtime to mimic the environment of the healing wound and thus should helpimprove wound outcomes. Another phenomenon observed duringre-epithelialization is increased expression of the integrin subunit ay.There is evidence that integrin, a major extracellular matrix receptor,polarizes in response to applied ES and thus controls directional cellmigration. It may be noted that there are a number of integrin subunits,however we chose integrin av because of evidence of association of avintegrin with IGF-1R, modulation of IGF-1 receptor signaling, and ofdriving keratinocyte locomotion. Additionally, integrin_(αv) has beenreported to contain vicinal thiols that provide site for redoxactivation of function of these integrins and therefore the increase inprotein thiols that we observe under the effect of ES may be the drivingforce behind increased integrin mediated cell migration. Other possibleintegrins which may be playing a role in LLEC-induced IGF-1R mediatedkeratinocyte migration are α5 integrin and α6 integrin.

Materials and Methods

Cell culture—Immortalized HaCaT human keratinocytes were grown inDulbecco's low-glucose modified Eagle's medium (Life Technologies,Gaithersburg, Md., U.S.A.) supplemented with 10% fetal bovine serum, 100U/ml penicillin, and 100 μg/ml streptomycin. The cells were maintainedin a standard culture incubator with humidified air containing 5% CO₂ at37° C.

Scratch assay—A cell migration assay was performed using culture inserts(IBIDI®, Verona, Wisc.) according to the manufacturer's instructions.Cell migration was measured using time-lapse phase-contrast microscopyfollowing withdrawal of the insert. Images were analyzed using theAxioVision Rel 4.8 software.

N-Acetyl Cysteine Treatment—Cells were pretreated with 5 mM of the thiolantioxidant N-acetylcysteine (Sigma) for 1 h before start of the scratchassay.

IGF-1R inhibition—When applicable, cells were preincubated with 50 nMIGF-1R inhibitor, picropodophyllin (Calbiochem, Mass.) just prior to theScratch Assay.

Cellular H₂O₂ Analysis—To determine intracellular H₂O₂ levels, HaCaTcells were incubated with 5 μM PF6-AM in PBS for 20 min at roomtemperature. After loading, cells were washed twice to remove excess dyeand visualized using a Zeiss Axiovert 200M microscope.

Catalase gene delivery—HaCaT cells were transfected with 2.3×10⁷ pfuAdCatalase or with the empty vector as control in 750 μL of media.Subsequently, 750 μL of additional media was added 4 h later and thecells were incubated for 72 h.

RTK Phosphorylation Assay—H uman Phospho-Receptor Tyrosine Kinasephosphorylation was measured using Phospho-RTK Array kit (R & DSystems).

ELISA—Phosphorylated and total IGF-1R were measured using a DuoSet ICELISA kit from R&D Systems.

Determination of Mitochondrial Membrane Potential—Mitochondrial membranepotential was measured in HaCaT cells exposed to the LLEC or placebousing TMRM or JC-1 (MitoProbe JC-1 Assay Kit for Flow Cytometry, LifeTechnologies), per manufacturer's instructions for flow cytometry.

Integrin aV Expression—Human HaCaT cells were grown under the MCD orplacebo and harvested 6 h after removing the IBIDI® insert. Staining wasdone using antibody against integrin aV (Abcam, Cambridge, Mass.).

Example 4 Wound Care Study

The medical histories of patients who received “standard-of-care” woundtreatment (“SOC”; n=20), or treatment with an LLEC device as disclosedherein (n=18), were reviewed. The wound care device used in the presentstudy consisted of a discrete matrix of silver and zinc dots. Asustained voltage of approximately 0.8 V was generated between the dots.The electric field generated at the device surface was measured to be0.2-1.0 V, 10-50 μA.

Wounds were assessed until closed or healed. The number of days to woundclosure and the rate of wound volume reduction were compared. Patientstreated with LLEC received one application of the device each week, ormore frequently in the presence of excessive wound exudate, inconjunction with appropriate wound care management. The LLEC was keptmoist by saturating with normal saline or conductive hydrogel.Adjunctive therapies (such as negative pressure wound therapy [NPWT],etc.) were administered with SOC or with the use of LLEC unlesscontraindicated. The SOC group received the standard of care appropriateto the wound, for example antimicrobial dressings, barrier creams,alginates, silver dressings, absorptive foam dressings, hydrogel,enzymatic debridement ointment, NPWT, etc. Etiology-specific care wasadministered on a case-by-case basis. Dressings were applied at weeklyintervals or more. The SOC and LLEC groups did not differ significantlyin gender, age, wound types or the length, width, and area of theirwounds.

Wound dimensions were recorded at the beginning of the treatment, aswell as interim and final patient visits. Wound dimensions, includinglength (L), width (W) and depth (D) were measured, with depth measuredat the deepest point. Wound closure progression was also documentedthrough digital photography. Determining the area of the wound wasperformed using the length and width measurements of the wound surfacearea.

Closure was defined as 100% epithelialization with visible effacement ofthe wound. Wounds were assessed 1 week post-closure to ensure continuedprogress toward healing during its maturation and remodeling phase.

Wound types included in this study were diverse in etiology anddimensions, thus the time to heal for wounds was distributed over a widerange (9-124 days for SOC, and 3-44 days for the LLEC group).Additionally, the patients often had multiple co-morbidities, includingdiabetes, renal disease, and hypertension. The average number of days towound closure was 36.25 (SD=28.89) for the SOC group and 19.78(SD=14.45) for the LLEC group, p=0.036. On average, the wounds in theLLEC treatment group attained closure 45.43% earlier than those in theSOC group.

Based on the volume calculated, some wounds improved persistently whileothers first increased in size before improving. The SOC and the LLECgroups were compared to each other in terms of the number of instanceswhen the dimensions of the patient wounds increased (i.e., woundtreatment outcome degraded). In the SOC group, 10 wounds (50% for n=20)became larger during at least one measurement interval, whereas 3 wounds(16.7% for n=18) became larger in the LLEC group (p=0.018). Overall,wounds in both groups responded positively. Response to treatment wasobserved to be slower during the initial phase, but was observed toimprove as time progressed.

The LLEC wound treatment group demonstrated on average a 45.4% fasterclosure rate as compared to the SOC group. Wounds receiving SOC weremore likely to follow a “waxing-and-waning” progression in wound closurecompared to wounds in the LLEC treatment group.

Compared to localized SOC treatments for wounds, the LLEC (1) reduceswound closure time, (2) has a steeper wound closure trajectory, and (3)has a more robust wound healing trend with fewer incidence of increasedwound dimensions during the course of healing.

Example 5 Induction of Pre-Angiogenic Responses in Vascular EndothelialCells by Signaling Through VEGF Receptors Materials and Methods CellCultures and Reagents

Tissue culture reagents were obtained from Life Technologies UK. TheVEGFR inhibitor (catalog number 676475), the P13K inhibitor LY294002(catalog number 440202), the Akt inhibitor (catalog number 124005) andthe Rho kinase inhibitor Y27632 (catalog number 688001) were allobtained from Calbiochem. Rhodamine-phalloidin (E3478) was obtained fromMolecular Probes (Leiden, The Netherlands) and anti-tubulin conjugatedwith FITC was obtained from Sigma. The HUVEC cell line from ATCC wasused prior to passage 10. Dulbecco's modified Eagle's medium (DMEM) with10% fetal bovine serum (FBS) was used for culture cells and EF exposureexperiments.

Electric Field Stimulation

HUVEC cells were seeded in a trough formed by two parallel (1 cm apart)strips of glass coverslip (No. 1, length of 22 mm) fixed to the base ofthe dish with silicone grease. Scratch lines were made perpendicular tothe long axis of the chamber with a fine sterile needle and used asreference marks for directed cell migration. Cells were incubated for24-48 hours (37° C., 5% CO₂) before a roof coverslip was applied andsealed with silicone grease. The final dimensions of the chamber,through which current was passed, were 22×10×0.2 mm. Agar-salt bridgesnot less than 15 cm long were used to connect silver/silver-chlorideelectrodes in beakers of Steinberg's solution (58 mM NaCl, 0.67 mM KCl,0.44 mM Ca(NO₃)₂, 1.3 mM MgSO₄, 4.6 mM Trizma base, pH 7.8-8.0), topools of excess culture medium at either side of the chamber. Fieldstrengths were measured directly at the beginning of, the end of andduring each experiment. No fluctuations in field strength were observed.For drug inhibition experiments, cells were incubated with the VEGFRinhibitor 4-[(4′-chloro-2′-fluoro)phenylamino]-6,7-dimethoxyquinazoline(50 μM), the PI3K inhibitor LY294002 (50 μM), an Akt inhibitor1-L-6-hydroxymethyl-chiro-inositol2-[(R)-2-O-methyl-3-O-octadecylcarbonate] (50 μM), the Rho kinaseinhibitor Y27632 (50 μM), both Akt and Rho kinase inhibitors (10 μMeach) or latrunculin (50 nM) for 1 hour before EF stimulation. The sameconcentration of drug was present during EF exposure in a CO₂ incubator.

Quantification of Cell Behavior

A series of images was taken with an image analyser immediately beforeEF exposure and at 4, 8 and 24 hours of EF exposure. Cell orientationwas quantified as an orientation index (Oi), which is defined asOi=cos2(α), where α is the angle formed by the long axis of a cell witha line drawn perpendicular to the field lines. A cell with its long axisparallel to the vector of the EF will have an Oi of −1, and a cell withits long axis exactly perpendicular to the EF vector will have an Oi of+1. A randomly oriented population of cells will have an average Oi{defined as [Σ_(n)cos2(α)]÷n} of 0.The significance of thistwo-dimensional orientation distribution against randomness wascalculated using Rayleigh's distribution. A long:short axis ratio wascalculated for assessment of elongation.

Mean migration rate and directedness were quantified over 4 hoursbecause cells multiplied during longer EF exposures, making it difficultto define a clear migration path. The angle (θ) that each cell movedwith respect to the imposed EF vector was measured. The cos(θ)(directedness) is +1, if the cell moved directly along the field linestoward the cathode, 0 if the cell moved perpendicular to the EF vectorand −1 if the cell moved directly towards the positive pole. Averagingthe cosines {[Σ_(i)cos(θ)]÷N, where N is the total number of cells}yields an average directedness of cell movement.

A commercially available VEGF165 ELISA kit was obtained from R and D(Minneapolis, Min.), and the detailed technical instructions werefollowed. Confocal microscopy was as described. Statistical analyseswere performed using unpaired, two-tailed Student's t-test. Data areexpressed as mean ±s.e.m.

Results

Cells cultured without exposure to the EF had the typical cobblestonemorphology, with the long axis of the cell body oriented randomly. Incontrast, endothelial cells cultured in DC EFs underwent areorientation, with their long axis coming to lie perpendicular to thevector of the applied EF. This elongation and alignment in an applied EFresembles the response of endothelial cells to fluid shear stress.

Cell alignment was quantified using an orientation index Oi=cos2(α),where α is the angle formed between the long axis of a cell and a linedrawn perpendicular to the field lines. In cells oriented perpendicularto the field vector, the Oi is +1, cells parallel to the field vectorgive an Oi of −1 and random orientation gives an Oi of 0. We comparedthe elongation and reorientation of single cells with those of cells inmonolayers. They were broadly similar, with single cells respondingquicker and showing a significantly higher Oi (0.56±0.04, n=245) at 4hours of EF exposure than cells in a monolayer sheet (0.35±0.03, n=528).Both single cells and cells in monolayers, however, had a similar Oi by8 hours (0.71±0.03, n=227 and 0.62±0.03, n=312, respectively).

The perpendicular orientation of endothelial cells showed both time andvoltage dependency. Significant orientation was observed as early as 4hours after the onset of the EF. A steady increase of Oi indicatesgradually increasing perpendicular orientation with continued exposure.Longer EF exposure, up to 3 days at 100 mV mm⁻¹ (1 mV across a cell 10μm wide), induced striking orientation and elongation. EF exposure didnot induce any detrimental effects on the cells, which were perfectlyhealthy for up to 3-4 days in EFs.

Voltage dependency was more obvious at later times, with a higher Oi forcells cultured at higher voltages. After 24 hours at 300 mV mm⁻¹, almostall the cells were perpendicular. An EF strength as low as 75 mV mm⁻¹induced significant perpendicular orientation, with Oi of 0.19(significantly different from random orientation, p=4.4×10⁻⁶, n=433),whereas an EF of 50 mV mm⁻¹ did not. The threshold field strengthinducing perpendicular orientation of the endothelial cells wastherefore between 50 mV mm⁻¹ and 75 mV mm⁻¹. This is low, representingonly 0.5-0.75 mV across a cell with a diameter of 10 μm.

Reorientation of endothelial cells in EFs requires VEGFR activation

VEGF activation is a pivotal element in angiogenic responses andenhanced angiogenesis by electric stimulation in vivo is mediatedthrough VEGFR activation. To test whether EF-induced endothelial cellorientation might involve VEGF signaling, we quantified levels of VEGF.EF exposure (200 mV mm⁻¹, the same as that measured at skin wounds)significantly enhanced levels of VEGF released into the culture medium.Marked elevation of VEGF in the culture medium was observed as early as5 minutes after onset of the EF; this was reduced at 1 hour and 2 hours,rose again at 4 hours, and reached a high level by 24 hours.

Inhibition of VEGFR activation by inhibiting both VEGFR-1 and VEGFR-2with the drug4-[(4′-chloro-2′-fluoro)phenylamino]-6,7-dimethoxyquinazoline completelyabolished the reorientation of cells in an EF. This drug is a potentVEGFR inhibitor that inhibits the receptor tyrosine kinase activity (50%inhibitory concentrations of 2.0 μM and 100 nM for VEGFR-1 and VEGFR-2,respectively). It is very selective for VEGFR-1 and VEGFR-2 tyrosinekinase activity compared with that associated with the epidermal growthfactor (EGF) receptor (50-fold and 3800-fold, respectively). Themorphology of the cells treated with VEGFR inhibitor was very similar tocontrol cells. Cells still elongated, although their long axis wasslightly reduced, but they were oriented randomly. Inhibition of VEGFRscould conceivably have had detrimental effects on the long-termviability of cells and this could have influenced their orientationresponses. To test for this, we compared the orientation response aftera short period of inhibitor and EF application. The orientation responsewas completely abolished at 4 hours and 8 hours in an EF after VEGFRinhibition. The Oi values of the cells treated with VEGFR inhibitor were−0.16±0.05 and −0.05±0.05 in EF for 4 hours and 8 hours, respectively,which is significantly different from the non-inhibitor-treated valuesof 0.36±0.05 and 0.53±0.05 (P<0.01).

Reorientation of endothelial cells involved the PI3K-Akt pathway

VEGFR activation lead to endothelial cell migration, cell survival andproliferation, which require the activation of Akt, a downstreameffectors of PI3K. Both the PI3K inhibitor LY294002 (50 μM) and the Aktinhibitor (50 μM) significantly decreased the orientation response.

The concentration of either drug alone would be expected to inhibit PI3Kand Akt activation completely but neither drug inhibited perpendicularreorientation completely, and significant Oi values remained, indicatingthat other signaling mechanisms must be involved.

Role of Rho and integrin in EF-induced reorientation of endothelialcells

The Rho family of GTPases regulates VEGF-stimulated endothelial cellmotility and reorganization of the actin cytoskeleton, which areimportant in endothelial cell retraction and in the formation ofintercellular gaps. The Rho kinase inhibitor, Y27632, decreased theorientation response significantly, with Oi values of 0.55±0.05,0.45±0.05 and 0.24±0.05 at 10 μM, 20 μM and 50 μM, respectively.Significant Oi values nonetheless remained even at 50 μM, indicatingthat multiple signaling mechanisms must be involved.Mitogen-activated-protein kinase inhibition with U0126 (50 μM), likeY27632 (0.33±0.03), decreased the orientation to a similar extent.

Because both Akt and Rho kinase inhibitors individually showed partialinhibition, perhaps the two enzymes function in different pathways toinduce cell reorientation. To test this, a combination of the twoinhibitors was used. The orientation response was abolished completelyby using Akt and Rho kinase inhibitors together (both at 10 μM)(Oi=−0.10±0.06; compared to control=0.80±0.09, P<0.0001).

Integrins, especially αvβ3, are important in endothelial cell movementand alignment to shear stress and mechanical stimulation. HUVEC cellswere incubated with a blocking antibody against αvβ3 (LM609) (20 μgml⁻¹) for 1 hour and then exposed to an EF (200 mV mm⁻1) with theantibody present. Blocking αvβ3 had no effect on orientation to the EF,cells reoriented normally (Oi=0.72±0.03, n=110, compared with thecontrol=0.80±0.09, n=124, P>0.05).

Small EFs Elongated Endothelial Cells.

HUVEC cells elongated dramatically in an EF. By contrast, cells culturedwith no EF retained a more-cobblestone-like appearance. Striking cellelongation was induced by a voltage drop of about 0.7-4.0 mV across acell of ˜15 μm in diameter. We quantified the elongation of the cellsusing a long:short axis ratio. A perfectly round cell has a long:shortaxis ratio of 1 and, as cells elongate, the ratio increases. Controlcells (no EF) showed no increase in long:short axis over 24 hours inculture. Elongation responses were both time and voltage dependent. Thelong:short axis ratio of EF exposed cells indicated gradual cellelongation throughout the 24 hour experimental period. The voltagedependency of the elongation response was more obvious at later times,with a greater long:short axis ratio for cells cultured at higher EFs.The threshold for EF-induced endothelial cell elongation was between50-75 mV mm⁻¹, again 0.5-0.75 mV across a cell 10 μm in diameter. Theelongation response of endothelial cells was more marked than that seenpreviously at the same EF strengths, in corneal and lens epithelialcells.

VEGFR, PI3K-Akt and Rho signaling are involved in the elongationresponse.

The signaling elements required for reorientation are also involved inelongation, but there are subtle differences. The VEGFR inhibitor (50μM) had no effect on the long:short axis ratio of control cells butsignificantly decreased the long:short axis ratio in EF-treated cells(P<0.002). Both the PI3K inhibitor LY294002 and the Akt inhibitor alsosignificantly decreased the long:short axis ratio (both P<0.0001 versuscontrol). Cells treated with these drugs elongated less, with LY294002the more effective in suppressing EF-induced elongation. The Rho kinaseinhibitor, Y27632 also significantly decreased the long:short axis ratio(P<0.0001), whereas the αvβ3-blocking antibody significantly inhibitedthe elongation response (3.12±0.008 compared with the control 3.65±0.15,P=0.007).

Cytoskeleton Alignment and the Consequence of Actin Filament Disruption.

To control changes in cell shape, reorientation and migration,extracellular stimuli initiate intracellular signaling that modifiescytoskeletal organization. Both actin filaments and microtubules werealigned in the direction of cell elongation. Latrunculin A, a toxininhibiting actin polymerization, completely abolished the EF-inducedelongation response and suppressed the orientation responsesignificantly (P<0.001) but not fully.

Small EFs direct migration of endothelial cells towards the anode.

Endothelial cells migrated directionally toward the anode when culturedin EFs. The directional migration was slow but steady during the EFexposure and was more evident for single cells than for sheets of cells.Cells migrated directionally towards the anode while elongating andreorienting perpendicularly. Lamellipodial extension toward the anodewas marked. Directional migration was obvious at a physiological EFstrength of 100 mV mm⁻¹. The threshold field strength that could inducedirectional migration was therefore below 100 mV mm⁻¹. Cell migrationwas quantified as previously and significant anodal migration wasevident (P<0.0001). Migration speed, however, remained constant beforeand after EF exposure, at 1-2 μm hour¹, which is significantly slowerthan most other cell types migrating in an EF.

Example 6 Effect on Propionibacterium acnes Bacterial Strains andCulture

The main bacterial strain used in this study is Propionibacterium acnesand multiple antibiotics-resistant P. acnes isolates are to beevaluated.

ATCC medium (7 Actinomyces broth) (BD) and/or ATCC medium (593 choppedmeat medium) is used for culturing P. acnes under an anaerobic conditionat 37° C. All experiments are performed under anaerobic conditions.

Culture

LNA (Leeming-Notman agar) medium is prepared and cultured at 34° C. for14 days.

Planktonic Cells

P. acnes is a relatively slow-growing, typically aero-tolerantanaerobic, Gram-positive bacterium (rod). P. acnes is cultured underanaerobic condition to determine for efficacy of an embodiment disclosedherein (PROCELLERA®). Overnight bacterial cultures are diluted withfresh culture medium supplemented with 0.1% sodium thioglycolate in PBSto 10⁵ colony forming units (CFUs). Next, the bacterial suspensions (0.5mL of about 105) are applied directly on PROCELLERA® (2″×2″) and controlfabrics in Petri-dishes under anaerobic conditions. After 0 h and 24 hpost treatments at 37° C., portions of the sample fabrics are placedinto anaerobic diluents and vigorously shaken by vortexing for 2 min.The suspensions are diluted serially and plated onto anaerobic platesunder an anaerobic condition. After 24 h incubation, the survivingcolonies are counted.

Bacterial Biofilms in Skin Infections

It is generally accepted that many human infections are biofilm-relatedand that sessile (biofilm-grown) cells are highly resistant againstantimicrobial agents. It has been suggested that P. acnes cells residingwithin the follicles grow as a biofilm. P. acnes readily forms biofilmsin vitro as well as on various medical devices in vivo, combined withthe high resistance of sessile P. acnes cells and the increasedproduction of particular virulence factors.

Example 7 Modulation of Bacterial Gene Expression and Enzyme Activity

Treatment of biofilms presents a major challenge, because bacterialiving within them enjoy increased protection against host immuneresponses and are markedly more tolerant to antibiotics. Bacteriaresiding within biofilms are encapsulated in an extracellular matrix,consisting of several components including polysaccharides, proteins andDNA which acts as a diffusion barrier between embedded bacteria and theenvironment thus retarding penetration of antibacterial agents.Additionally, due to limited nutrient accessibility, thebiofilm-residing bacteria are in a physiological state of low metabolismand dormancy increasing their resistance towards antibiotic agents.

Chronic wounds present an increasing socio-economic problem and anestimated 1-2% of western population suffers from chronic ulcers andapproximately 2-4% of the national healthcare budget in developedcountries is spent on treatment and complications due to chronic wounds.The incidence of non-healing wounds is expected to rise as a naturalconsequence of longer lifespan and progressive changes in lifestyle likeobesity, diabetes, and cardiovascular disease. Non-healing skin ulcersare often infected by biofilms. Multiple bacterial species reside inchronic wounds; with Pseudomonas aeruginosa, especially in largerwounds, being the most common. P. aeruginosa is suspected to delayhealing of leg ulcers. Also, surgical success with split graft skintransplantation and overall healing rate of chronic venous ulcers ispresumably reduced when there is clinical infection by P. aeruginosa.

P. aeruginosa biofilm is often associated with chronic wound infection.The BED (“BED” or “bioelectric device” or PROCELLERA® as disclosedherein) consists of a matrix of silver-zinc coupled biocompatiblemicrocells, which in the presence of conductive wound exudate activatesto generate an electric field (0.3-0.9V). Growth (measured as O.D andcfu) of pathogenic Pseudomonas aeruginosa strain PAO1 in LB media wasmarkedly arrested in the presence of the BED (p<0.05, n=4). PAO1 biofilmwas developed in vitro using a polycarbonate filter model. Grownovernight in LB medium at 37° C. bacteria were cultured on sterilepolycarbonate membrane filters placed on LB agar plates and allowed toform a mature biofilm for 48 h. The biofilm was then exposed to BED orplacebo for the following 24 h. Structural characterization usingscanning electron microscopy demonstrated that the BED markedlydisrupted biofilm integrity as compared to no significant effectobserved using a commercial silver dressing commonly used for woundcare. Staining of extracellular polymeric substance, PAO1 staining, anda vital stain demonstrated a decrease in biofilm thickness and number oflive bacterial cells in the presence of BED (n=4). BED repressed theexpression of quorum sensing genes lasR and rhIR (p<0.05, n=3). BED wasalso found to generate micromolar amounts of superoxide (n=3), which areknown reductants and repress genes of the redox sensing multidrug effluxsystem mexAB and mexEF (n=3, p<0.05). BED also down-regulated theactivity of glycerol-3-phosphate dehydrogenase, an electric fieldsensitive enzyme responsible for bacterial respiration, glycolysis, andphospholipid biosynthesis (p<0.05, n=3).

Materials and Methods In-vitro Biofilm Model

PAO1 biofilm was developed in vitro using a polycarbonate filter model.Cells were grown overnight in LB medium at 37° C. bacteria were culturedon sterile polycarbonate membrane filters placed on LB agar plates andallowed to form a mature biofilm for 48 h. The biofilm was then exposedto BED or placebo for the following 24 h.

Energy Dispersive X-ray Spectroscopy (EDS)

EDS elemental analysis of the Ag/ZN BED was performed in anenvironmental scanning electron microscope (ESEM, FEI XL-30) at 25 kV. Athin layer of carbon was evaporated onto the surface of the dressing toincrease the conductivity.

Scanning Electron Microscopy

Biofilm was grown on circular membranes and was then fixed in a 4%formaldehyde/2% glutaraldehyde solution for 48 hours at 4° C., washedwith phosphate-buffered saline solution buffer, dehydrated in a gradedethanol series, critical point dried, and mounted on an aluminum stub.The samples were then sputter coated with platinum (Pt) and imaged withthe SEM operating at 5 kV in the secondary electron mode (XL 30S; FEG,FEI Co., Hillsboro, Oreg.).

Live/Dead Staining

The LIVE/DEAD BacLight Bacterial Viability Kit for microscopy andquantitative assays was used to monitor the viability of bacterialpopulations. Cells with a compromised membrane that are considered to bedead or dying stain red, whereas cells with an intact membrane staingreen.

EPR Spectroscopy

EPR measurements were performed at room temperature using a Bruker ER300 EPR spectrometer operating at X-band with a TM 110 cavity. Themicrowave frequency was measured with an EIP Model 575 source-lockingmicrowave counter (EIP Microwave, Inc., San Jose, Calif.). Theinstrument settings used in the spin trapping experiments were asfollows: modulation amplitude, 0.32 G; time constant, 0.16 s; scan time,60 s; modulation frequency, 100 kHz; microwave power, 20 mW; microwavefrequency, 9.76 GHz. The samples were placed in a quartz EPR flat cell,and spectra were recorded at ambient temperature (25° C.). Serial 1-minEPR acquisitions were performed. The components of the spectra wereidentified, simulated, and quantitated as reported. The double integralsof DEPMPO experimental spectra were compared with those of a 1 mM TEMPOsample measured under identical settings to estimate the concentrationof superoxide adduct.

Quantification of mRNA and miRNA Expression

Total RNA, including the miRNA fraction, was isolated using Norgen RNAisolation kit, according to the manufacturer's protocol. Gene expressionlevels were quantified with real-time PCR system and SYBR Green (AppliedBiosystems) and normalized to nadB and proC as housekeeping genes.Expression levels were quantified employing the 2 (−ΔΔct) relativequantification method.

Glycerol-3-Phosphate Dehydrogenase Assay

The glycerol-3-phosphate dehydrogenase assay was performed using anassay kit from Biovision, Inc. following manufacturer's instructions.Briefly, cells (˜1×10⁶) were homogenized with 200 μl ice cold GPDH Assaybuffer for 10 minutes on ice and the supernatant was used to measureO.D. and GPDH activity calculated from the results.

Statistics

Control and treated samples were compared by paired t test. Student's ttest was used for all other comparison of difference between means.P<0.05 was considered significant.

Aq/Zn BED Disrupts P. aeruginosa Biofilm

To validate the chemical composition of the dressing, we collected highresolution electron micrographs using an environmental scanning electronmicroscope. Our element maps indicate that silver particles areconcentrated in the golden dots of the polyester cloth, while zincparticles are concentrated in the grey dots.

As illustrated in FIG. 14A, P. aeruginosa was grown in round bottomtubes in LB medium with continuous shaking and absorbance was measuredby calculating optical density at 600 nm at different time points. Itwas observed that Ag/Zn BED and the control dressing with equal amountof silver inhibited bacterial growth (n=4) (FIG. 14B,C). When bacteriais grown in an agar plate with Ag/Zn BED dressing or placebo embedded inthe agar, the zone of inhibition is clearly visible in the case of Ag/ZnBED thus demonstrating its bacteriostatic property, while placebo withsilver dressing showed a smaller zone of inhibition, indicating theeffect role of electric field as compared to topical contact. (FIG.14D). However, as evident from scanning electron microscope images (FIG.15); EPS staining (FIG. 16); and live/dead staining (FIG. 17), Ag/Zn BEDdisrupts biofilm much better while silver does not have any effect onbiofilm disruption. Silver has been recognized for its antimicrobialproperties for centuries. Most studies on the antibacterial efficacy ofsilver, with particular emphasis on wound healing, have been performedon planktonic bacteria. Silver ions, bind to and react with proteins andenzymes, thereby causing structural changes in the bacterial cell walland membranes, leading to cellular disintegration and death of thebacterium. Silver also binds to bacterial DNA and RNA, therebyinhibiting the basal life processes.

Silver is effective against mature biofilms of P. aeruginosa, but onlyat a high silver concentration. A concentration of 5-10 μg/mL silversulfadiazine has been reported to eradicate biofilm whereas a lowerconcentration (1 μg/mL) had no effect. Therefore, the concentration ofsilver in currently available wound dressings is much too low fortreatment of chronic biofilm wounds. FIG. 18 shows PAO1 staining of thebiofilm demonstrating the lack of elevated mushroom like structures inthe Ag/Zn BED treated sample.

Aq/Zn BED Down-Requlates Quorum Sensing Genes

The pathogenicity of P. aeruginosa is attributable to an arsenal ofvirulence factors. The production of many of these extracellularvirulence factors occurs only when the bacterial cell density hasreached a threshold (quorum). Quorum sensing is controlled primarily bytwo cell-to-cell signaling systems, called las and rhl, which are bothcomposed of a transcriptional regulator (LasR and RhIR, respectively)and an autoinducer synthase (LasI and RhII, respectively). In P.aeruginosa, LasI produces 3OC12-HSL. LasR, then, responds to this signaland the LasR:3OC12-HSL complex activates transcription of many genesincluding rhIR, which encodes a second quorum sensing receptor, RhIRwhich binds to autoinducer C4-HSL produced by RhII. RhIR:C4-HSL alsodirects a large regulon of genes. P. aeruginosa defective in QS iscompromised in their ability to form biofilms. Quorum sensing inhibitorsincrease the susceptibility of the biofilms to multiple types ofantibiotics.

To test the effect of the electric field on quorum sensing genes, wesubjected the mature biofilm to the Ag/Zen BED or placebo for 12 hoursand looked at gene expression levels. We selected an earlier time point,because by 24 hours, as in earlier experiments, most bacteria underAg/Zn BED treatment were dead. We found a significant down regulation oflasR and rhIR (n=4, p<0.05). lasR transcription has been reported toweakly correlate with the transcription of lasA, lasB, toxA and aprA. Wedid not, however, find any significant difference in their expressionlevels at this time point, although we found them down regulated in theAg/Zn BED treated samples at the 24 hour time point (data not shown).(FIG. 19).

Aq/Zn BED Represses the Redox Sensing Multidrug Efflux System in P.aeruginosa

Ag/Zn BED acts as a reducing agent and reduces protein thiols. Oneelectron reduction of dioxygen O₂, results in the production ofsuperoxide anion. Molecular oxygen (dioxygen) contains two unpairedelectrons. The addition of a second electron fills one of its twodegenerate molecular orbitals, generating a charged ionic species withsingle unpaired electrons that exhibit paramagnetism. Superoxide anion,which can act as a biological reductant and can reduce disulfide bonds,is finally converted to hydrogen peroxide is known to have bactericidalproperties. Here, we used electron paramagnetic resonance (EPR) todetect superoxide directly upon exposure to the bioelectric dressing.Superoxide spin trap was carried out using DEPMPO(2-(diethoxyphosphoryl)-2-methyl-3,4-dihydro-2H-pyrrole 1- oxide) and ˜1μM superoxide anion production was detected upon 40 mins of exposure toAg/Zn BED (FIG. 20). MexR and MexT are two multidrug efflux regulatorsin P. aeruginosa which uses an oxidation-sensing mechanism. Oxidation ofboth MexR and MexT results in formation of intermolecular disulfidebonds, which activates them, leading to dissociation from promoter DNAand de-repression of MexAB-oprM and MexEF-oprN respectively, while in areduced state, they do not transcribe the operons. Induction of Mexoperons leads not only to increased antibiotic resistance but also torepression of the quorum sensing cascades and several virulence factors.We observe down-regulation of the downstream Mex genes MexA, MexB, MexEand MexF (but not MexC and MexD) (n=4, p<0.05), in Ag/Zn BED treatedsamples, inactive forms of MexR and MexT in their reduced states. Toconfirm the reducing activity of the Ag/Zn BED, the experiments wererepeated with 10 mM DTT and similar results were observed. (FIG. 21).

Aq/Zn BED Diminishes Glycerol-3-Phosphate Dehydrogenase Enzyme Activity

Electric fields can affect molecular charge distributions on manyenzymes. Glycerol-3-phosphate dehydrogenase is an enzyme involved inrespiration, glycolysis, and phospholipid biosynthesis and is expectedto be influenced by external electric fields in P. aeruginosa. Weobserved significantly diminished glycerol-3-phosphate dehydrogenaseenzyme activity by treating P. aeruginosa biofilm to the Ag/Zn BED for12 hours (n=3). (FIG. 22).

Example 8 LLEC Influence on Biofilm Properties

In this study ten clinical wound pathogens associated with chronic woundinfections were used for evaluating the anti-biofilm properties of anLLEC. Hydrogel and drip-flow reactor (DFR) biofilm models were employedfor the efficacy evaluation of the wound dressing in inhibitingbiofilms. Biofilms formed with Acinetobacter baumannii, Corynebacteriumamycolatum, Escherichia coli, Enterobacter aerogenes, Enterococcusfaecalis CI 4413, Klebsiella pneumonia, Pseudomonas aeruginosa, Serratiamarcescens, Staphylococcus aureus, and Streptococcus equi clinicalisolates were evaluated. For antimicrobial susceptibility testing ofbiofilms, 10⁵ CFU/mL bacteria was used in both biofilm models. Forpoloxamer hydrogel model, the LLECs (25 mm diameter) were applieddirectly onto the bacterial biofilm developed onto 30% poloxamerhydrogel and Muller-Hinton agar plates, and incubated at 37° C. for 24 hto observe any growth inhibition. In the DFR biofilm model, bacteriawere deposited onto polycarbonate membrane as abiotic surface, andsample dressings were applied onto the membrane. The DFR biofilm wasincubated in diluted trypticase soy broth (TSB) at room temperature for72 h. Biofilm formations were evaluated by crystal violet staining underlight microscopy, and anti-biofilm efficacy was demonstrated byreduction in bacterial numbers.

Example 9 Modulation of Mammalian Gene Expression and Enzyme Activity

Grown overnight in LB medium at 37° C., primary human dermal fibroblastsare cultured on sterile polycarbonate membrane filters placed on LB agarplates for 48 h. The cells are then exposed to BED or placebo for thefollowing 24 h. BED represses the expression of glyceraldehyde3-phosphate dehydrogenase. BED also down-regulates the activity ofglyceraldehyde 3-phosphate dehydrogenase.

Example 10 Modulation of Insect Gene Expression and Enzyme Activity

Grown overnight in LB medium at 37° C., drosophila S2 cells are culturedon sterile polycarbonate membrane filters placed on LB agar plates for48 h. The cells are then exposed to BED or placebo for the following 24h. BED represses the expression of insect P450 enzymes. BED alsodown-regulates the activity of insect P450 enzymes.

Example 11 Effect on Propionibacterium acnes

To determine the antimicrobial properties of the devices as disclosedherein, assessment of antibacterial finishes on textile materials wasconducted in accordance with the American Association of TextileChemists and Colorists (AATCC) Testing Methodology 100-1993. Themicrocurrent generating dressings were tested against multiple pathogensand successfully demonstrated antimicrobial properties against a broadspectrum of pathogens as shown in Table 1 below.

TABLE 1 In-vitro Percent Reduction in Microorganisms Microorganism %Reduction Escherichia coli 99.99 Aspergillus niger 99.27 Trichophytonmentagropytes 99.22 Vancomycin Resistant Enterococcus (VRE) 99.97Streptococcus pneumoniae 100 Methicillin Resistant Staph Aureus 100(MSRA) Staphylococcus aureus 100 Acinetobacter baumaanii 100Trichophyton rubrum 99.99 Corynebacterium xerosis 100 Trichophytonashii/inkin 99.97 Pseudomonas aeruginosa 100 Candida albicans 99.98Propionibacterium acnes 100 Klebsiella Pneumonaie 100 Herpes SimplexType 1 100 Varicella Virus 99.98

Further studies will be performed as described:

Outcome Measures

Primary outcomes: Change in acne severity grade from baseline andsplit-back comparison as determined by Leeds Acne Grading System11 andblinded clinician extender evaluation. Change in acne severity based onmasked photographic assessment

Secondary outcomes: Patient subjective outcomes, per user assessmentsurvey. Patient assessment of wearability of study device

Study DesignThis is a double-blinded, two-arm, same-patient, split-back,prospective study investigating 50 patients presenting with acnevulgaris on the back; Patients will serve as their own controls and weara vest as described herein comprising a substrate with biocompatibleelectrodes configured to generate an electric field, or in the presenceof a conductive solution, an electric current.

Study Site: This multicenter study will be conducted at two facilities;Paradise Valley Dermatology, Phoenix, Az. and Arizona AdvancedDermatology, Phoenix/Gilbert, Az.

Selection of Patients: The study population will include subjects age 14years 40 years.

Number of Patients: 50 subjects completing up to at least week 6 oftreatment will be considered evaluable. Subject population will includemale or female subjects of all ethnic groups. Parental consent fortreatment of minors will be obtained for subjects <18 years.

Inclusion Criteria:

-   -   a. Clinical diagnosis of mild or moderate acne vulgaris of the        back (Leeds scale grades 1-5)    -   b. Subjects age 14 years 40 years    -   c. Participants willing to undergo treatment and participate in        follow up evaluations. Participants willing to comply with study        procedures and willing to refrain from “picking” at lesions.    -   d. Participants with cell phones and willing to receive daily        text reminders to wear study device.    -   e. Participants falling outside the washout periods for topical        and systemic treatments and office procedures.    -   f. 4 weeks for topical agents on the back (corticosteroids,        retinoids and other acne treatments);    -   g. 8 weeks for office-based acne procedures on the back (for        chemical peeling, laser and light-based therapies).    -   h. 12 weeks for systemic drugs (corticosteroids and other acne        treatments)    -   i. Subjects requiring topical agents for the face or requiring        office-based acne procedures for the face will be included in        the study.    -   j. Participant must be able to read and understand informed        consent, and signs the informed consent

Exclusion Criteria

-   -   a. Clinical diagnosis of severe acne vulgaris of the back        requiring systemic drugs for management, as defined by Leeds        Scale Grades 6    -   b. Less than 14 years of age or over 40 years of age.    -   c. Patients receiving any topical or office-based acne        procedures within the washout period prior to study.    -   d. Participation in another clinical trial that involved the use        of an investigational drug or device that in the opinion of the        investigator would confound the results of this trial    -   e. Individuals with silver or zinc sensitivity    -   f. Active cancer    -   g. Immunosuppressive treatment    -   h. Clinically diagnosed hyperandrogenic state    -   i. Evidence of severe androgen excess (i.e., testosterone        levels >150 ng/dL)    -   j. Excessive back hair in male patients    -   k. Pregnant or nursing individuals    -   l. Patients with pacemakers    -   m. Cystic or nodular acne lesions    -   n. Use of medicated shampoos, body washes, exfoliants and benzyl        peroxide washes for 4 weeks prior to study start.    -   o. Any other medical condition in the clinician's opinion that        excludes the patient.    -   p. Geographical concerns (residence not within reasonable travel        distance) that would hamper compliance with required study        visits.    -   q. The Investigator believes that the subject will be unwilling        or unable to comply with study protocol requirements, including        application of study device and all study-related follow up        visit requirements.

The participants must answer “yes” on all inclusion criteria and mustanswer “no” on all exclusion criteria

Patient Recruitment

Patients treated at Paradise Valley Dermatology, Arizona AdvancedDermatology, Center for Dermatology, and the Skin and Cancer Center ofAZ presented with acne vulgaris of the back will be given theopportunity to enroll into the study. At time of screening, thephysician extender will document non-identifiers including date, sex,age, presence of back acne, and interest in participation in a study. Ifthere is an interest, the patients will be given a detailed explanationof the risks and benefits of the study.

Method of Obtaining Informed Consent

Written informed consent will be obtained from the patients by thephysicians, residents and/or staff present in the clinic who aretreating the individual patients

Plan of Study

The system to be used in the present study is a 2-part vest sdesigned tocover the scapular area of the back. The outer vest is comprised ofvinyl, polyester trim and elastic materials and adjustable straps. Thevest contains a detachable absorbent pad comprised of polyvinyl acetate(PVA) lined with the multi-array matrix of biocompatible microcellsprinted on one half of the vest, and a placebo pattern on the other halfof the vest

Description of Study Procedures Randomization

Patients will be randomized into Group X or Group Y. 25 patients will berandomized to receive vests with the multi-array matrix of biocompatiblemicrocells printed on the right half and placebo pattern on the lefthalf (Group X) and 25 patients will receive vests with multi-arraymatrix of biocompatible microcells printed on the left half and placeboon the right half (Group Y). The patient, investigators and physicianextenders (nurses, PA's, etc) will be blinded to the assigned side ofthe vest. Just prior to dissemination of study materials, theinvestigator will be provided with the randomization of treatment theparticipant will receive in the duration of the study, Group X or GroupY. A randomization key will be held at a secure location with the studysponsor. After final study analysis, the key will be revealed. In theevent the randomization is made apparent, a note will be documented inthe patient file. Patient will still be included in the study and studydata will still be considered evaluable.

Group Treatment Vest Configuration

Group X Multi-array matrix of biocompatible microcells on RIGHT side ofvest and placebo pattern on LEFT side of vest

Group Y Multi-array matrix of biocompatible microcells on LEFT side ofvest and plascebo pattern on RIGHT side of vest

Study Materials

Participants will be provided the following study supplies:

-   -   a. Plastic vest replacements    -   b. Removable multi-array matrix of biocompatible microcells vest        pad replacements    -   c. Supply of compression shirts    -   d. Study Log    -   e. Carrying tote for materials

Participants will be instructed in the use of the multi-array matrix ofbiocompatible microcells vest, and will be supplied with detailedinstructions for use.

Device Application Method

Before going to bed, patient will be instructed to apply the vestembodiment according to the provided instructions. Patients will moistenthe pad of the vest and secure the vest to the body. They will wear aspandex t-shirt covering the entire vest. They will continue to wear thevest throughout the course of the night and remove the nighttime vest inthe morning after awakening.

Patient Compliance Monitoring

Patients will be reminded to wear their study device by documenting wearthrough a paper log and/or electronic technology platform.

Automated Patient Communications

An automated patient communication portal, i.e. Constant Contact,SolutionReach, PracticeMojo, etc. will be used for patient compliancepurposes. During the consent process, will have agreed to participate inautomated text reminders. To remind patients to wear the study deviceeach day, patients will receive a daily evening reminder on theircellular phone to wear the study device, as well as periodic remindersthroughout the week (˜3 times week) to change the vest pads. In the2-way communication platform, participants will be requested to text “C”for confirming compliance. To ensure patient confidentiality andprivacy, all text communications will comply with all applicable rulesand regulations—see Appendix 1.

Deviations:

Participants will be asked to document all deviations from studyprocedures, and log dates where the vest was not worn due to extenuatingcircumstances.

-   Study Period    -   a. Visit 0—Baseline Visit 1—Week 2    -   b. (Day 14±2), Visit 2—Week 4 (Day 28±2), Visit 3—Week 6    -   c. (Day 42±2).-   Study Enrollment-   Consent form/Minor Consent-   Medical History-   Baseline Acne Grading-   Digital Photos-   Skin Evaluation-   Vest distribution-   Standard Follow-up Procedures:-   Digital Photos-   Skin Evaluation-   Acne Grading-   Vest distribution-   Standard Follow-up Procedures-   Digital Photos-   Skin Evaluation-   Acne Grading-   Vest distribution-   Standard Follow-up Procedures-   Digital Photos-   Skin Evaluation-   Acne Grading-   End of Study-   Clinic Visits    -   a. Patients will be seen at the clinic for a total of four        visits during the study, with baseline visit 0 serving as the        initial visit. Follow-up visits will occur when the participants        return to the clinic at weeks 2, 4 and 6 after the initial        baseline visit for their follow-up evaluations.-   Clinic Assessments-   Initial Intake

Basic patient demographic information will be collected at the initialstudy visit, including:

-   -   a. Age    -   b. Gender    -   c. Past acne treatments    -   d. Other skin conditions and/or relevant medical conditions

The following study procedures will be performed at each visit:

Clinician Acne Grading: At the initial and each follow-up visit, theacne lesions will assessed by a physician extender, who will be blindedto the patients' randomization. Five separate evaluations will beperformed at each visit and will be documented on case report form.

-   -   a. 1) Leeds Rating: Grading of acne severity will be conducted        according to the Leeds Acne Grading System, an overall        assessment of acne severity for use in routine clinic., which        grades patients on a scale of 0 (no acne) to 10 (the most        severe). For the purpose of this study, only patients falling in        between grades 1-5 will be included.

2) Clinical Evaluation

-   -   a. A standard skin assessment, evaluating presence of infection,        erythema, and irritation will be documented.

3) Lesion Count

-   -   a. The area of the back above the scapula will be divided into        four equal quadrants:

Left upper, left lower, right upper and right lower. The physicianextender will count and document the number of lesions observed in eachquadrant.

4) Global Assessment

-   -   a. Efficacy of each treatment will be investigated by global        assessment; the physician extender will assess if the global        acne appearance on each half of the back has improved, is the        same, or has worsened compared to the baseline photo.

5) Side by Side Assessment

-   -   a. The physician extender will perform a “side-by-side”        evaluation to evaluate the appearance of acne of the left side        compared to the right side of the back, and document if the left        or right side of the back looks better, or the same.

Photograph

-   -   a. Standardized digital photography to capture progressive        changes in acne lesions over time will be performed at each        visit. A standard photography station will be set up at each        clinic site, with digital camera mounted on tripod. The back        will be photographed according to the following standards:    -   b. Angle: The photo is to be taken perpendicular to the back    -   c. Lighting: The photo is to be taken under the same lighting        conditions.    -   d. Height: The photo is to be taken at a standard height for        each patient, with height adjustable as needed.    -   e. Necessary information to accompany each photo: Label with        participant # and date    -   f. Number of photos: Photos will be obtained at each follow-up        visit; a total of 4 photographs will be taken: Baseline, Week 2,        Week 4, and Week 6 of study.    -   g. Pre and post-treatment photos will be overlaid for        comparison.

Patient Acne Assessment

-   -   a. At the follow-up visits, patients will evaluate photos of        their back at the current study visit and self-report on the        following: general appearance of acne, redness and discomfort of        acne lesions.    -   b. Patient clinical assessment will be documented on case report        form “Patient Acne Assessment” XV-060CRF-06.

Wearability Assessment

-   -   a. Participants will also answer a questionnaire at their last        visit to assess their experience with the vest. Participants        will rate comfort level (vest, strap, moisture, temperature,        sleep quality) and other observations during the study. Outcomes        will be captured on “Wearability assessment” XV-060CRF-08.

Blinded Clinician Photo Assessment

-   -   a. Photos will be graded per by two independent clinicians, with        a third clinician brought in if tie-breaking is required.        Clinicians will be provided digital photos of each participant,        with photos out of chronologic order

In the first series of four photos, clinicians will perform a “side byside” comparison evaluating left side of back versus right side of backin each of the photos, and selecting which side appears better, Left-Right- or if they appear the Same.

In the second series of three photos, clinicians will perform a globalassessment evaluating left half of back versus baseline photo, and righthalf of back versus baseline photo. Baseline photo is labeled butsubsequent photos are not in chronologic order.

Study Endpoint

The study will end 6 weeks after first day of enrollment in the study.All patients will be monitored for adverse side effects including butnot limited to infection, skin sensitivity, and worsening condition ofthe acne condition during the course of the study.

Study Outcomes: All patients will be assessed for:

-   -   a. Acne improvement via Leeds System and clinical skin        assessment    -   b. Visual acne improvement via digital photography    -   c. Wearability assessment via patient survey    -   d. Patient self assessment of acne clinical condition

Example 12 Preventing Viral Propagation

A mammal infected with MERS wears a disclosed “insert” embodiment insidean neck gaiter to prevent viral propagation. The insert is moistenedwith saline prior to use.

Example 13 Preventing Viral Propagation

A mammal infected with a corona virus wears a disclosed neck gaiter toprevent viral propagation. The neck gaiter is moistened with salineprior to use.

Example 14 Preventing Viral Acquisition

A healthy mammal wears a disclosed neck gaiter embodiment to preventviral acquisition.

Example 15 Preventing Viral Acquisition

A healthy mammal wears a disclosed neck gaiter embodiment to preventviral acquisition.

Example 16 Viral Proliferation Test

A disclosed embodiment was tested against several viral strains.According to the results, there was 100% kill after a 10⁴ PFU viralchallenge/sample.

Virus Kind Influenza Virus Feline Calcivirus Virus Influenza A Virus(H3N2) Feline calicivirus Strain Influenza A Virus (H1N1) Strain: F-9Host Cell MCDK Cell (Dog kidney cell CRFK Cell (Cat kidney origin) cellorigin)

Results # of Plaques of Fabrics (Vomaris) Time Point Blank Zinc SilverProcellera Notes T30 TMTC TMTC 0 0 T60 TMTC TMTC 0 0 TMTC, too many tocount plaques

The antiviral effects were observed within minutes of exposure, as shownin FIG. 27 and FIG. 28.

Example 17 Coronavirus Test

Electroceutical Fabric for PPE against COVID-19

Abbreviations: COVID-19—Coronavirus Disease of 2019; CDC—Center forDisease Control 23 and Prevention; PPE—personal protective equipment;SEM—Scanning electron microscopy; CoV 24—Coronavirus; SARS—Severe acuterespiratory syndrome; CPE—Cytopathic effects; WHO—25 World HealthOrganization; FDA—Food and Drug Administration; NTA—Nanoparticletracking 26 analysis; EDX—Energy Dispersive X-ray microanalysis;kV—kilovolt; keV—kilo electronvolt; 27 ddH2O—ultra-pure water; GFP—greenfluorescent protein; MTT-3-(4,5-dimethylthiazol-2-yl)-28 2,5-diphenyltetrazolium bromide

In this work we tested the hypothesis that an electroceutical fabricwill disrupt the infectivity of coronavirus upon contact bydestabilizing the electrokinetic properties of the viral particle. Arespiratory coronavirus (USDA permit 141794) and the correspondingmammalian ST cell were obtained from ATCC to study the cytopathiceffects of viral infection. Viral particles (105) were placed in directcontact with the electroceutical or sham fabric for either 1 or 5minutes. Viral particles (4×10⁴) were recovered from the fabric andsubjected to nanoparticle tracking analysis and measurement of zetapotential. Recovered viral particles were subjected to cytopathictesting and studied for 7 days following infection. Under conditions ofcytopathic testing, the electroceutical fabric generated a weakpotential difference of 0.5V. Following one minute of contact, zetapotential of the coronavirus was significantly lowered indicatingdestabilization of its electrokinetic properties. Size-distribution plotshowed appearance of aggregation of the virus. Testing of the cytopathiceffects of the virus showed eradication of infectivity as quantitativelyassessed by calcein-PI and MTT cell viability tests. This work presentsfirst evidence demonstrating that the physical characteristic featuresof CoV may be exploited to render it non-infective following exposure toweak electric field generating electroceutical fabric. The effect israpid and achieved within one minute of contact. The supportingobservation that lentiviral infectivity is also eliminated followingcontact with the electroceutical fabric contributes to the rigor of ourcentral finding.

Results Characterization of the Electroceutical Fabric

The electroceutical fabric tested is made up of polyester fabric printedwith alternating circular regions of Ag and Zn dots. Ag dots (2 mm) andZn dots (1mm) were printed on the fabric in proximity of about 1 mm toeach other. Scanning electron microscopy (SEM) displayed the depositionof Ag particles and Zn on the fibers of the polyester fabric. EnergyDispersive X-ray (EDX) microanalysis revealed the presence of Ag and Znon the electroceutical fabric (fe) and absence in the sham polyesterfabric (fs). The only peak that was present other than C and O was thatof Au used for coating the fabrics for SEM imaging. Proximity of Ag andZn on polyester fabric forms a redox couple and is capable of drivingelectrochemistry when wet in an aqueous ionized environment includingany body fluid. Ag and Zn were spotted on another textile which was alsoappropriate for the preparation of stretchable face-masks. SEM of thefabric used for such mask showed a different weaving pattern aimed athigher stretch property. Deposition of Ag and Zn on the fabric forface-mask was tested by EDX spectrum analysis. Our primary line ofinvestigation focused on the polyester-based electroceutical fabricwhich may be utilized for the development of PPE as well. Three ionizedaqueous media were used to test potential difference between adjacent Agand Zn deposits. NaCl solution (0.85% w/v), cell culture medium and tapwater (of practical value to end users of PPE) were tested at roomtemperature. The potential difference between the two electrodes rapidlyincreased and achieved a steady state after the first 15 s.

Physical Characterization of the Coronavirus

SEM (150,000x) revealed the morphological features of the CoV particle.Following spotting on the silicon wafer, the purified virus was fixedand subsequently dehydrated. A thin (2-132 5 nm) layer of carbon wassputtered on the sample to make the specimen conductive. The size of thevirus ranged between 75-125nm. Nanoparticle tracking analysis (NTA)revealed poly-134 dispersed peak. The electrokinetic property, asrepresented by the zeta potential, of the viral particles is a parameterthat determines adsorption and stability of the particle in any givendispersant medium. For practical purposes, viral particles are expectedto be suspended in water droplets either aerosolized or resting on asurface. The average zeta potential of four different preparation of CoVwas determined to be −25.675 mV. All four-preparation demonstratedcomparable zeta potential distribution and phase shift. The averageelectrophoretic mobility distribution was determined to be −2 μmcm/Vs.

Electroceutical Fabric Attenuated the zeta Potential of Coronavirus UponContact

Quantification of the purified viral particles after spotting on feyielded 44.29% and 23.73% recovery from the fabric when exposed for 1min or 5 min, respect ively (FIG. 29A). Nanoparticle tracking analysisdemonstrated that unlike the purified CoV that showed a single peakaround 75 nm, the recovered CoV showed additional peaks suggestingaggregation of the viral particles upon contact with the fabric (FIG.29B). Analysis of □ potential showed significant graded attenuation ofthis electrokinetic property upon contact with the fe (FIG. 29C). Suchlowering of average zeta potential of CoV, applied and recovered fromfe, has been plotted graphically (FIG. 29D). Unlike 1 min exposure tothe fe, 5 min exposure showed an appreciable difference in the phaseplot of the viral particles (FIG. 29E).

Loss of Corona Virus Infectivity upon Contact with ElectroceuticalFabric

To assess changes in the infectivity of CoV following contact with theelectroceutical fabric, a cytopathic assay was employed. Infected cellswere monitored for appearance of cytopathic effects (CPE; cell roundingand sloughing) until post-infection day 7. Overt CPE was observed on day7 in response to CoV infection (FIG. 30B; FIG. 33). Comparable CPE wasnoted in response to treatment of cells with CoV recovered from shamcontrol fabric fs (FIG.30C; FIG. 33). In contrast, CoV recovered from fedid not cause any CPE indicating loss of its infectivity (FIG. 30D; FIG.S2). Cells treated with fe-recovered CoV particles appeared as healthyas the uninfected cells (FIG. 30A; FIG. 33). Objective assessment ofcell viability was performed using a calcein/PI fluorescence assay. Onlylive cells with intracellular esterase activity hydrolyze theacetoxymethyl ester in non-fluorescent Calcein AM converting it intogreen fluorescent Calcein. Dead cells or cells with damaged orcompromised cell membranes include PI stain, which is otherwiseimpermeant to live cells. Fold-change increase in PI/Calcein signal asshown indicates loss of cell viability in response to infection.Infection of cells with CoV caused marked loss of cell viability (FIG.30B). Such cytopathic effect of CoV was completely absent once the viruswas exposed to fe (FIG. 30 D-E). The sham fabric did not afford suchprotection (FIG. 30C,E). The cytopathic effects of CoV and theprotective effects of fe (versus fs) was corroborated by the standardMTT assay commonly used for testing cell viability (FIG. 30F).

Electroceutical Fabric Eliminated Lentiviral Transduction Efficacy

The Lentiviral pseudotype system is a standard laboratory tool to studythe infectivity of viruses under conventional biosafety conditions.Lentivirus CSCGW mut6, upon successful transduction in HEK293 cells,results in GFP-expressing host cells. This expression is a directmeasure of lentiviral replication competency and ability of the virus tointegrate in the host genome. The ability of the electroceutical fabricto influence the infectivity of a virus, other than CoV, was tested toappreciate its broader significance of scope. Mammalian cells weretreated with purified lentivirus or the same virus subjected to contactwith fe or fs for 1 or 5 mins as indicated in the figure legend (FIG.31). Transduced cells were monitored microscopically to check thepresence of GFP+cells, a marker of successful infection. Lentiviralexposure caused widespread infection of cells. Treatment of cells withvirus recovered from sham fabric fs caused comparable infection (FIG.31B). However, contact of virus with the electroceutical fabric fe, evenfor one minute, eliminated lentiviral infectivity (FIG. 31B).

Discussion

Previous work from our laboratory has established the effectiveness ofelectroceutical principles as an alternative to pharmacologicalapproaches in managing planktonic microbial pathogens and complexpolymicrobial biofilms. The currently studied electroceutical fabric issimple in configuration not requiring any complex wiring or powersource. It is thus easy to use in a field setting and requires notraining or skills. The textile itself is comparable to any otherstandard textile lending itself for manufacturing of PPE. Viruses areknown to rely on electrostatic interactions for optimal virion assemblyand attachment. For instance, structural proteins in coronaviruses,negatively charged amino acid residues in the nucleocapsid facilitatesassembly with the membrane protein. Additionally, the coronavirusenvelope protein is known to generate ion conductive pores acrossmembranes which are voltage dependent 23. Leveraging these viralcharacteristics to achieve viral inactivation remains largely unexploredand has been attempted in this work.

Electroceuticals have generated renewed interest in the health careindustry. This work presents the first evidence demonstrating that theinfectivity of the CoV may be disrupted using a simple electroceuticalfabric. The fabric tested in this work consists of only silver and zincdots on polyester fabric that forms a redox couple. This workdemonstrates that CoV infectivity may be rapidly eradicated upon contactwith the electroceutical fabric. Zeta potential of a particle determinesits electrostatic interactions in particle dispersions and, as such, isan important determinant of the stability of viral particles. Contact ofCoV with the electroceutical fabric studied rapidly lowered the zetapotential demonstrating a direct effect of the fabric on theelectrokinetic properties of the viral particle. Any change of zetapotential towards zero is viewed as increase in electrical instabilityof the particle. The observation that contact with the electroceuticalfabric eliminates infectivity of the virus leads to the hypothesis thatthe observed lowering of zeta potential may have caused defects in thestructural integrity of the virus. Study of changes in the capsid-RNAstructure following exposure to the weak electric field generated by thefabric is thus warranted. CoV is a nanoparticle. Nanoparticle trackinganalysis determines the hydrodynamic diameter of the analyte by applyingthe Stokes-Einstein equation after measuring the Brownian motion ofindividual nanoparticle. It is an alternative method to dynamic lightscattering which utilizes the same principle and is validated forassessing polydispersity and purity in viral vaccine preparations. NTAwas therefore utilized to estimate absolute viral particle number andsize distribution in not only pure CoV but also in CoV recovered fromthe fabric. Observed changes in particle number and size distributionsupport the aforementioned hypothesis that exposure to the weak electricfield causes damaging structural alterations to the virions. Cells inculture routinely display a small fraction of dead or dying cells.Cytopathic effects of viral infection are tested to examine whetherexposure to the infectious particle adds to the basal cell death burdenof the culture. Long-term observations, i.e. days versus hours, ensurethe recording of the eventual fate of the affected cells. Reporting ofshort-term data alone, while sometimes may be encouraging with respectto effect of the intervention, may simply reflect results representingpostponement of death from the insult and not a true rescue. In CPEstudies of this work, cell rounding and sloughing were evident in day 4post-infection. During this time, cells treated with virus pre-exposedto the electroceutical fabric closely resembled cells that wereunchallenged by exposure to the virus. In standard cell culture, thegrowth medium is changed every other day to wash off floating dead cellsand to replenish nutrition. Under conditions of infection by virus, suchfrequent change of cell culture medium is not made. Cells grow in thesame spent media until day 7 post-infection. Maintenance of cellswithout any change in culture media for seven days is expected tomarginally increase basal cell death burden as shown.

Textiles evaluated for use in PPE such as masks are subject to specificFDA 510(k) requirements expecting stringent viral filtration tests todemonstrate 99.9% reduction of 1.1-3.3×10⁴ plaque forming units ofstandard phiX174 bacteriophage. The phiX174 is widely used as a modelorganism because of it being a standardized test. However, it isimportant to note that unlike SAR-241 CoV-2 which is a RNA virus,phiX174 bacteriophage is a DNA virus with numerous contrasting physical,chemical as well as biological properties. Furthermore, thisbacteriophage is much smaller in size than SAR-CoV-2. The non-envelopedicosahedral morphology of phiX174 bacteriophage aerosolizes with a meanparticle size of 3.0±0.3 μm25. This is in direct contrast with thecoronaviruses that cause diseases in animals and humans which are ˜100nm in diameter and are aerosolized as respiratory droplets with sizes >5μm. Importantly, phiX174 cannot and are aerosolized as respiratorydroplets with sizes >5 μm. Importantly, phiX174 cannot infect mammaliancells. It infects and forms visible plaques on a lawn of Escherichiacoli (Migula) Castellani and Chalmers strains. In the context ofCOVID-19 pandemic, our study focuses on coronavirus and tests cytopathiceffects on mammalian cells. Testing methods such as AATCC 250 TM100recommends a textile contact time of 24 h for both enveloped andnon-enveloped viruses. We report results on contact time that is muchshorter and more relevant to PPE usage in the context of COVID-19.Additional studies in our laboratory show effective neutr alization of awider range of viruses at a much higher load (108) within 1-2 h oftextile contact time (not shown).

This work presents first evidence demonstrating that the physicalcharacteristic features of CoV may be exploited to render itnon-infective following exposure to weak electric field generatingelectroceutical fabric. The effect is rapid and achieved within oneminute of contact. The observation that lentiviral infectivity is alsoeliminated following contact with the electroceutical fabric contributesto the rigor of our central finding. Lowering of zeta potential of theCoV particles following exposure to the electroceutical fabricconstitutes direct evidence supporting the contention thatelectrokinetic stability of the viral particle is weakened. Additionalstudies are necessary to characterize specific structural changes inresponse to exposure to the electroceutical fabric, and to connect suchchanges to loss of infectivity. In the meanwhile, this work providescompelling first evidence to consider the studied electroceuticalfabric, or other materials with similar property, as material of choicefor the development of PPE in the fight against COVID-19.

Materials and Methods Electroceutical Fabric 2

An FDA cleared wireless electroceutical dressing was used as a source ofweak electric field for the current study and is referred to aselectroceutical fabric (fe). This fabric, co-developed by ourlaboratory, has been commercialized by Vomaris Inc. (Phoenix, Az.) wasprovided to us by the manufacturer. It is made of polyester fabricprinted with alternating circular dots of Ag and Zn metals (2 mm and 1mm, respectively), generating electric fields. A polyester fabricwithout any metal deposition (hence unable to generate electric field)was used as an experimental control and is referred to as sham fabric(fs).

Viruses and Cell Lines

Respiratory coronavirus (ATCC® VR2384™) and its host porcine cellline—ST (ATCC® CRL-278 1746™), recommended for its infection andpropagation were procured from ATCC (Manassas, Va.).

Cell Culture

Cell lines were cultured and maintained in respective cell culturemedium, in either T25 or T75 flasks (Cat no: 82051-074 and 82050-856,Greiner Bio-One, Monroe, N.C.), at 37° C. and humidified 5% CO2 in airatmosphere. All culture media were made complete by addition of FetalBovine Serum (FBS, final concentration 10%; Cat no: F2442-500ML,Sigma-Aldrich, St. Louis, Mo.) and Antibiotic-Antimycotic solution(final concentration 1X; Cat no: 15240-062, Life Technologies, Carlsbad,Calif.). For coronavirus studies, ST cells were cultured in completeEagle's Minimum Essential Medium (EMEM, ATCC® 30-2003™). HEK293 cellswere cultured in complete Dulbecco's Modified Eagle's Medium (DMEM, Catno: 11995073, Gibco™, Gaithersburg, Md.). For sub-culturing HEK293cells, culture medium was discarded from flasks and cells (85-90%confluent) were rinsed briefly with 5 ml of 1X phosphate buffered saline(PBS; Cat no: 20012027, Gibco™, Gaithersburg, Md.) to remove all tracesof serum. Cells were detached by adding of 2-3 ml of 0.05% Trypsin-EDTAsolution (Cat no: 25300054, Gibco™, Gaithersburg, Md.) and incubation at37° C. for 15 minutes. Respective complete growth medium (˜4-6 ml) wasadded to the flasks. Detached cells were aspirated with gentle pipettingand this cell suspension was centrifuged at 500×g for 3 mins at 28° C.(Beckman Coulter Allegra x-14r-SX4750). Post centrifugation, supernatantwas discarded, and cells were thoroughly re-suspended in either 5 ml(for T25 flask) or 12 ml (for T75 flask) of complete growth mediumfollowed by addition in new culture flasks and incubation as mentionedearlier. For cryopreservation, cells were trypsinized and pelleted asabove to re-suspend in 2 ml of complete growth medium with 5% (v/v)dimethyl sulfoxide (Cat no: BP231-100, Fisher Scientific, Waltham,Mass.). These cells were first stored at −20° C. for 3 h, followed bystorage at −80° C. for 24 h and final storage in liquid nitrogen.

Respiratory Coronavirus (Coy) Infection and Propagation

ST cells were cultured in complete EMEM till they attained a confluencyof 80-90% followed by washing monolayers with 5 ml of 1X PBS. USDApermit 141794 (Dr. Sen) was obtained for the procurement and laboratoryuse of coronavirus. Coronavirus stock (ATCC VR-2384) was thawed at 37°for 5 min and aliquots of 250 μl were prepared for further use orstorage in liquid nitrogen.

An aliquot of this stock was diluted with 3 ml of incomplete culturemedium (without FBS and Antibiotic-Antimycotic solution) to attain aMultiplicity of Infection (MOI) of 1 as per ATCC recommendations. Thisdiluted viral stock was added to the washed monolayer and incubated at37° C., humidified 5% CO2 in air atmosphere. Flasks were rocked gentlyfor 2 min at intervals of 315 30 min, to re-distribute viral inoculum.Post 2 h infection, viral adsorption was ended by adding 10 ml ofcomplete culture medium to the monolayer. Cells were monitoredmicroscopically every 24 h for signs of cytopathic effects (CPE. Flasksshowing CPE in 80% of the infected host cells were used for viralpurification.

Coronavirus Purification

The protocol adopted for viral purification was as per Maier et al.,201529. Culture medium from flasks with infected cells was harvested at10000×g for 20 mins at 4° C. (Beckman Coulter Allegra x-14r-FX6100).Viral precipitation from this supernatant (12 ml) was done by additionof polyethylene glycol (PEG-6000, final concentration 10%; Cat no:81260, Sigma-Aldrich, St. Louis, Mo.) and NaCl (final concentration2.2%; Cat no: S271, Fisher Scientific, Waltham, Mass.).PEG-6000/NaCl/culture supernatant mixture was incubated at 4° C. for 30minutes. PEG precipitated proteins and virions were pelleted at 10000×gfor 30 min at 4° C. and the pellet was dissolved in 100 μl of ice-cold1X HEPES-saline buffer (10 mM HEPES—Sigma H7523+0.9% w/v NaCl, pH 6.7).Dissolved pellet was then loaded on a discontinuous sucrose gradient(10-20-30%, 800 μl each; freshly prepared in 1X HEPES-saline) andsubjected to ultracentrifugation at 100000×g for 90 mins at 4° C.(Beckman Coulter Optima MAX-XP Ultracentrifuge). Postultra-centrifugation, the supernatant was discarded, and the viralpellet was dissolved in 100 μl of 1X HEPES-saline buffer (pH 7.4). Totalviral particle number estimation was performed using 17

Nanoparticle Tracking Analysis (NTA) and purified viruses were flashfrozen in dry ice followed by storage in liquid nitrogen, till furtheruse.

Nanoparticle Tracking Analysis

Viruses were diluted in EMEM (ATCC® 30-2003™) or 18.2 MΩ water. Meanparticle diameter and concentration of viral particles were analyzed byNanoSight NS300 with a 532 nm laser and SCMOS camera (Malvern,Worcestershire, UK) as previously described30 31. Briefly, samples werediluted 100:1 or as needed in fresh milliQ to obtain 5-100particles/frame. Samples were typically analyzed using 5 runs of 30 scollecting 25 frames per second (749 frames per run) with viscositydetermined by the temperature and camera level highest available forsample (typically 15 or 14). The syringe pump speed was 60. NTAautomatically compensates for flow in the sample such that only Brownianmotion is used for size determination. For processing results, thedetection threshold was typically 5 with automated blur size and maxjump distance. Standard 100 nm latex spheres were run at 1000:1 dilutionin milliQ to test optimal instrument performance. Data were analyzedusung NTA 3.0 software (Malvern Instruments Ltd., UK).

Zeta Potential Analysis

Zeta potential measurement of viral particles was determined byZetasizer (Nano-Z, Malvern Instruments Ltd., UK) as describedpreviously30, 31. All samples were dispersed in double-distilled waterand tested in volume-weighted size distribution mode in folded capillarycells (Fisher Scientific NC0491866). An average of three readings (60 s)were recorded.

Scanning Electron Microscopy of CoV

Viral particles were suspended in ddH2O with 2.5% glutaraldehyde orother buffer and dropped onto dean silica wafers. After drying, sampleswere desiccated in a vacuum chamber for at least 12 h before -analysis.Images were obtained after carbon sputter coating using a field emissionscanning electron microscope (JEOL 7800F, JEOL Japan) at a beam energyof 5 or 10 kV. For the SEM images of the fabric, gold sputter coatingwas used.

Energy Dispersive X-ray Microanalysis

For elemental detection, the Energy Dispersive X-ray (EDX) microanalysisassociated to scanning electron microscopy was used. When the electronbeam hits the gold sputtered fabric, some atoms of the sample areexcited or ionized. When excited or ionized atom return to their groundstate, they emit characteristic x-rays. The x-ray emissions at differentwavelengths were measured using a photon-energy-sensitive detector. TheEDX detector system performs a simultaneous display of all mid-energy(1-20 keV) x-rays collected during any individual analysis period.

Coronavirus and Lentivirus Infectivity

ST (coronavirus) and HEK 293 (lentivirus) cells were seeded at densitiesof 10,000/well and 1000/well in 24-well and 96-well cell culture plates,respectively. Seeded plates were incubated at 37° C., 5% CO₂ humidifiedincubator for 18 h. One hundred microliter (105 particles) of aqueoussuspension of viruses (106/ml of VR-2384 and CSCGW mut6 lentivirus) werespotted on 1.5 cm diameter discs of fe and fs at room temperature. Afteran incubation period of 1 min or 5 min, 100 μl of serum free medium wasused to rinse each fabric for recovering viral particles from thefabric. NTA was performed, as above, to estimate viral recoveryefficiency.

Recovered VR-2384 viruses were diluted with serum free medium and usedto infect ST cells at MOI of 10 (105 viruses). Recovered CSCGW mut6virus was diluted in complete DMEM medium followed by HEK293transduction at MOI of 10 (4×104 viruses). Parallel sets of cellsinfected with untreated viruses (at the same MOI as that of treatedviruses) were used as positive control while uninfected ornon-transduced host cells were accounted as negative control. Virusinfected ST cells were monitored microscopically at intervals of 24 hfor the onset and progression of cytopathic effects. The expression ofGFP in transduced HEK293 was assessed after 4 days to ascertain theeffect of fe treatment on lentiviral infectivity. Six technicalreplicates were assayed for each experimental group. Twelve biologicalreplicates were studied.

Cell Viability Staining by Calcein AM and Propidium Iodide

Viability of ST cells, infected as above, was assessed by dual stainingwith Calcein AM and Propidium iodide (PI). Media from wells with STcells (uninfected or infected with untreated or fabric-contactedviruses) was washed briefly with 1 ml of 1X PBS (per well) for 1 min,followed by addition of 250 μl of freshly-prepared staining solution in1X PBS (Calcein AM; final concentration 1 μg/ml, Catalog no: C1430,Invitrogen™, Waltham, Mass.) and PI (final concentration 10 μg/ml,Catalog no: AS 25535-16-4 Sigma-Aldrich, St. Louis, Mo.). Cells wereincubated under dark conditions at 37° C. for 15 min and then observedunder a Confocal Laser Scanning Microscope using a 10X objective.Multiple images (10 images per group/per set) were captured andfluorescence intensities were calculated from these images using Zenblue software and graphically plotted as shown. The ratio of PI:Calceinsignal was normalized with the average

PI intensity of untreated cells to obtain fold-change compared tonon-viable cells (basal cell death) in untreated cells.

Cell Viability Assessment by MTT Assay

Cell viability of ST cells infected as above was assayed using the MTT(344,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.This assay was performed as per manufacturer's protocol (MTT assay kit,Catalog no: ab211091, Abcam, Cambridge, Mass.). ST cells were washedwith 1 ml of 1X PBS per well and then harvested using a cell scraper.Cells were collected in tubes, centrifuged at 300×g for 5 min at roomtemperature followed by re-suspension in incomplete EMEM. Cells in thissuspension were counted and cell count in all the experimental groupswas normalized to 105 cells per 500 μl of culture medium. In separate96-well tissue culture plates, 50 μl of the above suspension was addedin each well (final cell counts as 104 cells). To each well, 50 μl ofMTT solution was added and the plates were incubated at 37° C., 5% CO2humidified incubator for 3 hours. After incubation, plates werecentrifuged at 300×g for 5 min at room temperature and supernatant wasdiscarded. One hundred and fifty microliters of MTT solvent was added toeach well. Plates were wrapped in aluminum foil and rocked on an orbitalshaker for 15 min followed by measurement of absorbance at 590 nm.

Statistical Analysis

GraphPad Prism (GraphPad Software) v8.0 was used for statisticalanalyses. Statistical analysis 419 between multiple groups wereperformed using one-way analysis of variance with the post hoc 420 Sidakmultiple comparison test. Statistical analysis between two groups wasperformed using unpaired Student's two-sided t tests. P<0.05 wasconsidered statistically significant. Significance levels and exact Pvalues are indicated in all relevant figures. Data were normallydistributed. Data for independent experiments were presented as means±SEM unless otherwise stated. Individual data points are plottedreflecting n (8-19) for each experiment.

Example 18 Neck Gaiter Insert

A user affixes via adhesive a disclosed neck gaiter insert to the insideof a reusable neck gaiter. After 6 hours, the insert is removed andreplaced with a new insert. The removed insert is washed and can bereused.

Example 19 Neck Gaiter Insert

A user affixes via adhesive a disclosed neck gaiter insert to theoutside of a reusable neck gaiter. After 6 hours, the insert is removedand replaced with a new insert. The removed insert is washed and can bereused.

Example 20 Neck Gaiter Insert

A user inserts a hydrated (with saline) insert into an envelope-slotinside of a reusable neck gaiter. After 7 hours, the insert is removedand replaced with a new insert. The removed insert is washed and can bereused.

Example 21 Neck Gaiter Insert

A user affixes with Velcro a hydrated neck gaiter insert to the insideof a reusable neck gaiter. After 8 hours, the insert is removed andreplaced with a new insert. The removed insert is washed and can bereused.

Example 22 Neck Gaiter Insert

A user affixes with Velcro a hydrated neck gaiter insert to the outsideof a reusable neck gaiter. After 8 hours, the insert is removed andreplaced with a new insert. The removed insert is washed and can bereused.

In closing, it is to be understood that although aspects of the presentspecification are highlighted by referring to specific embodiments, oneskilled in the art will readily appreciate that these disclosedembodiments are only illustrative of the principles of the subjectmatter disclosed herein. Therefore, it should be understood that thedisclosed subject matter is in no way limited to a particularmethodology, protocol, and/or reagent, etc., described herein. As such,various modifications or changes to or alternative configurations of thedisclosed subject matter can be made in accordance with the teachingsherein without departing from the spirit of the present specification.Lastly, the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present disclosure, which is defined solely by the claims.Accordingly, embodiments of the present disclosure are not limited tothose precisely as shown and described.

Certain embodiments are described herein, including the best mode knownto the inventor for carrying out the methods and devices describedherein. Of course, variations on these described embodiments will becomeapparent to those of ordinary skill in the art upon reading theforegoing description. Accordingly, this disclosure includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described embodiments in all possiblevariations thereof is encompassed by the disclosure unless otherwiseindicated herein or otherwise clearly contradicted by context.

Groupings of alternative embodiments, elements, or steps of the presentdisclosure are not to be construed as limitations. Each group member maybe referred to and claimed individually or in any combination with othergroup members disclosed herein. It is anticipated that one or moremembers of a group may be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is deemed to contain the group asmodified thus fulfilling the written description of all Markush groupsused in the appended claims.

Unless otherwise indicated, all numbers expressing a characteristic,item, quantity, parameter, property, term, and so forth used in thepresent specification and claims are to be understood as being modifiedin all instances by the term “about.” As used herein, the term “about”means that the characteristic, item, quantity, parameter, property, orterm so qualified encompasses a range of plus or minus ten percent aboveand below the value of the stated characteristic, item, quantity,parameter, property, or term. Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the specification andattached claims are approximations that may vary. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical indication shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and values setting forth the broad scope ofthe disclosure are approximations, the numerical ranges and values setforth in the specific examples are reported as precisely as possible.Any numerical range or value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Recitation of numerical ranges ofvalues herein is merely intended to serve as a shorthand method ofreferring individually to each separate numerical value falling withinthe range. Unless otherwise indicated herein, each individual value of anumerical range is incorporated into the present specification as if itwere individually recited herein.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the disclosure (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein is intended merely to better illuminate thedisclosure and does not pose a limitation on the scope otherwiseclaimed. No language in the present specification should be construed asindicating any non-claimed element essential to the practice ofembodiments disclosed herein.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the present disclosure so claimed areinherently or expressly described and enabled herein.

1. A system for preventing viral transmission through the mouth and nose comprising; a planar substrate comprising a long axis and further comprising one or more biocompatible electrodes configured to generate at least one of a low level electric field (LLEF) or low level electric current (LLEC), wherein said substrate comprises snaps, hooks, magnets, or velcro at opposite ends of its long axis.
 2. The system of claim 1 wherein the biocompatible electrodes comprise a first array comprising a pattern of microcells formed from a first conductive material, and a second array comprising a pattern of microcells formed from a second conductive material.
 3. The system of claim 2, wherein said planar substrate comprises a pouch to secure an insert to the planar substrate.
 4. The system of claim 2, wherein said planar substrate comprises a compression fabric.
 5. The system of claim 3 wherein said pouch comprises a flap to secure an insert to the planar substrate.
 6. The system of claim 2, wherein said planar substrate comprises a slot to secure an insert to the planar substrate.
 7. The system of claim 2, wherein said planar substrate comprises adhesive to secure an insert to the planar substrate.
 8. The system of claim 2 wherein said substrate is hydrated.
 9. The system of claim 1, further comprising a pouch for storing the system.
 10. A method for preventing viral transmission comprising applying the system of claim 2 to an area where prevention is desired.
 11. The method of claim 10, wherein said transmission comprises viral propagation.
 12. The method of claim 10, wherein said transmission comprises viral acquisition. 